Hybrizap - 2.1 Two-Hybrid Libraries: Instruction Manual

HybriZAP®-2.
1 Two-Hybrid
Libraries
INSTRUCTION MANUAL
Revision A
Manual #838401-13
For In Vitro Use Only

838401-13
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HybriZAP®-2.1 Two-Hybrid Libraries
CONTENTS
Materials Provided.............................................................................................................................. 1
Storage Conditions .............................................................................................................................. 1
Additional Materials Required .......................................................................................................... 2
Notice to Purchaser ............................................................................................................................. 2
Introduction......................................................................................................................................... 3
Overview of HybriZAP®-2.1 Two-Hybrid Library Screens.................................................. 4
Vectors.................................................................................................................................................. 5
HybriZAP®-2.1 Vector Map.................................................................................................. 6
pAD-GAL4-2.1 Vector Map ................................................................................................. 8
The pBD-GAL4 Cam Vector Map ........................................................................................ 9
Bacterial Host Strains ....................................................................................................................... 10
Bacterial Strain Genotypes .................................................................................................. 10
XL1-Blue MRF´ Bacterial Strain Description..................................................................... 10
Recommended Media.......................................................................................................... 11
Establishing an Agar Plate Bacterial Stock ......................................................................... 11
Preparation of a –80°C Bacterial Glycerol Stock................................................................ 11
Library Titer Determination ................................................................................................ 12
Yeast Host Strain .............................................................................................................................. 14
Yeast Strain Genotype and Phenotypic Verification ........................................................... 14
Yeast Strain Description...................................................................................................... 15
Preparation of the Yeast Host Strain ................................................................................... 16
Preparation of a –80°C Yeast Glycerol Stock ..................................................................... 16
Helper Phage ..................................................................................................................................... 17
Storing the Helper Phage..................................................................................................... 17
Titering the Helper Phage.................................................................................................... 17
Amplifying the Helper Phage .............................................................................................. 18
Control Plasmids ............................................................................................................................... 19
Description .......................................................................................................................... 19
Applications......................................................................................................................... 19
Expected Results for Control Plasmid Assays .................................................................... 22
®
In Vivo Excision of the pAD-GAL4-2.1 Phagemid Vector from the HybriZAP -2.1 Vector..... 23
ExAssist® Helper Phage and XLOLR Strain....................................................................... 24
Mass Excision Protocol ....................................................................................................... 24
Amplification of the Excised Phagemid Library ................................................................. 27
Single-Clone Excision Protocol .......................................................................................... 28
DNA-Binding Domain Vector Construction................................................................................... 30
Bait Protein Insert Preparation, Ligation, and Transformation ........................................... 30
Yeast Transformation and Assay for Expression of Reporter Genes .................................. 32
Yeast Transformation....................................................................................................................... 33
Simultaneous vs. Sequential Transformation of the Bait and Target Plasmids................... 33
Control Plasmids ................................................................................................................. 34
Yeast Transformation Protocol............................................................................................ 34
Screening............................................................................................................................................ 38
Filter Lift Assay................................................................................................................... 39
Verification of Interaction................................................................................................................ 40
Isolation of Plasmid DNA from Yeast ................................................................................ 40
Verification of Specificity of Protein–Protein Interactions ................................................. 43
Appendix: General Comparison of Escherichia coli versus Yeast Host Strains ......................... 44
Troubleshooting ................................................................................................................................ 45
Mass Excision...................................................................................................................... 45
Two-Hybrid Vector System Screening................................................................................ 45
Plasmid Isolation from Yeast .............................................................................................. 46
Verification of Interaction ................................................................................................... 47
Preparation of Media and Reagents ................................................................................................ 48
Standard Media and Reagents ............................................................................................. 48
Two-Hybrid Vector System Media and Reagents ............................................................... 50
References .......................................................................................................................................... 55
Endnotes............................................................................................................................................. 56
MSDS Information............................................................................................................................ 56
Note The complete sequences for the pAD-GAL4-2.1 phagemid vector and the pBD-GAL4 Cam
phagemid vector are available for downloading to your computer. The pAD-GAL4-2.1 and
pBD-GAL4 Cam phagemid vector sequences are available from www.stratagene.com or
from the GenBank® database (Accession #AF033313 and #U46126, respectively).
MATERIALS PROVIDED
Premade Library
Amplified premade library constructed in the HybriZAP-2.1 vector in 7% DMSO. On arrival,
store the premade library at –80°C. Do not pass through more than two freeze–thaw cycles.
HybriZAP®-2.1 Two-Hybrid cDNA Library Kit

Catalog #978000 (provided with Catalog #838401)
Material provided Quantity
pBD-GAL4 Cam phagemid vector 20 μg (1 μg/μl in TE buffer)
Control plasmids
pGAL4 control plasmid 20 μg (1 μg/μl in TE buffer)
pBD-WT control plasmid 20 μg (1 μg/μl in TE buffer)
pAD-WT control plasmid 20 μg (1 μg/μl in TE buffer)
pBD-MUT control plasmid 20 μg (1 μg/μl in TE buffer)
pAD-MUT control plasmid 20 μg (1 μg/μl in TE buffer)
pLamin C control plasmid 20 μg (1 μg/μl in TE buffer)
Bacterial host strains
XL1-Blue MRF´ strain 0.5-ml bacterial glycerol stock
XLOLR strain 0.5-ml bacterial glycerol stock
YRG-2 yeast host strain 1 ml
ExAssist® interference-resistant helper phagea ~1.0 × 1010 pfu/ml
STORAGE CONDITIONS
Control Plasmids: –20°C
Helper Phage: –80°C
Bacterial Glycerol Stocks: –80°C
YRG-2 Yeast Host Strain: –80°C
Premade Libraries: –80°C.
Revision A Copyright © 2006 by Stratagene.
HybriZAP®-2.1 Two-Hybrid Libraries 1

ADDITIONAL MATERIALS REQUIRED
Reagents and Solutions
Phenol–chloroform [1:1 (v/v)] and chloroform
Note Do not use the low-pH phenol from Stratagene’s RNA Isolation Kit for any
phenol–chloroform extractions within the yeast plasmid isolation protocol. The low-pH
phenol is specific for RNA isolation and may cause the DNA to remain in the organic
phase following extraction.
Salmon sperm DNA
Equipment and Supplies

Acid-washed glass beads (425–600 μm)
Wide-bore pipet tips
Water baths (4°, 12°, 30°, 37°, and 70°C)
Vacuum evaporator
Incubator (30° and 37°C)
14-ml BD Falcon™ polypropylene round-bottom tubes (BD Biosciences Catalog #352059)
Whatman® No. 1 qualitative filter paper, Grade 1 {Fisher Scientific, Pittsburgh, Pennsylvania
[Catalog #09-805C (7 cm diameter) and #09-805F (12.5 cm diameter)]}
VWRbrand™ qualitative filter papers, Grade No. 413 {VWR Scientific, Westchester, Pennsylvania
[Catalog #28310-026 (7.5 cm diameter)] and #28310-106 (12.5 cm diameter)]}
NOTICE TO PURCHASER
Practice of the two-hybrid system is covered by U.S. Patent Nos. 5,283,173; 5,468,614 and
5,667,973 assigned to The Research Foundation of State University of New York. Purchase of any
two-hybrid reagents does not imply or convey a license to practice the two-hybrid system covered by
these patents. Commercial entities in the U.S.A. practicing the above technologies must obtain a
license from The Research Foundation of State University of New York. Non-profit institutions may
obtain a complimentary license for research not sponsored by industry. Please contact Dr. John
Roberts, Associate Director, The Research Foundation of SUNY at Stony Brook, W5530 Melville
Memorial Library, Stony Brook, NY 11794-3368; phone 631 632 4163; fax 631 632 1505 for
license information.
The HybriZAP®-2.1 vector is covered by Stratagene's U.S. Patent Nos. 5,128,256 and 5,286,636.
Purchase of the HybriZAP-2.1 vector and/or the HybriZAP-2.1 vector systems does not grant rights
to (1) use the HybriZAP-2.1 vector for the reproduction, amplification, or modification of the vector;
(2) offer the HybriZAP-2.1 vector or any derivative thereof for resale; (3) distribute or transfer the
HybriZAP-2.1 vector or any derivative thereof to any third party; or (4) incorporate the
HybriZAP-2.1 vector or any derivative thereof in any genomic or cDNA library for resale,
distribution, or transfer to any third party.
2 HybriZAP®-2.1 Two-Hybrid Libraries

INTRODUCTION
Protein–protein interactions occur in many biological processes including
replication, transcription, secretion, signal transduction, and metabolism. A
fundamental question in the study of any protein is to identify proteins that
interact with a given protein in vivo. Intense research efforts are focused on
the identification of these proteins.
The HybriZAP®-2.1 two-hybrid vector system* (Figure 1), a eukaryotic

system to detect protein–protein interactions in vivo, provides a method for
the rapid identification of genes encoding proteins that interact with a given
protein (i.e., a bait protein).1, 2 The system is based on the ability to separate
eukaryotic transcriptional activators into two separate domains, the DNA-
binding domain (BD) and the transcriptional activation domain (AD).3 In the
HybriZAP-2.1 two-hybrid vector system, proteins that interact with the bait
protein are identified by generating hybrids of the yeast GAL4 BD and the
bait protein (X) and the GAL4 AD and a library of proteins (Y). Neither
hybrid protein is capable of initiating specific transcription of reporter genes
in yeast in the absence of a specific interaction with the other hybrid protein
(Figure 2A). When the hybrid protein X is expressed in yeast, the GAL4 BD
binds X to specific DNA sequences in the yeast chromosome defined by the
GAL1 or GAL4 upstream activating sequences (UASGAL1 or UASGAL4,
respectively), which regulate the expression of a reporter gene. Binding of X
to the UAS is not sufficient to initiate transcription of the reporter gene.
When Y is expressed in yeast, the AD interacts with other components of the
transcription machinery required to initiate transcription of the reporter
gene. However, Y alone is not localized to the reporter gene UAS and
therefore does not activate transcription of the reporter gene. When a
specific interaction between X and Y localizes both the GAL4 BD and
GAL4 AD to the reporter gene UAS, transcriptional activation of the
reporter gene occurs (Figure 2B). The reporter genes in the HybriZAP-2.1
two-hybrid vector system are β-galactosidase (lacZ) and histidine (HIS3).
The HybriZAP-2.1 two-hybrid vector system is particularly useful for the

identification of novel (target) proteins from a cDNA library, which interact
with a bait protein, and for the subsequent determination of protein domains
or amino acids critical for the interaction. Specific mutations, insertions, or
deletions that affect the encoded amino acid can be introduced into the
target protein, and the mutants can be assayed for the protein–protein
interaction with the bait protein.
* U.S. Patent Nos. 5,283,173; 5,468,614; 5,128,256; and 5,286,636.

Overview of HybriZAP®-2.1 Two-Hybrid Library Screens
pAD-GAL4-2.1
MCS
A-J att int xis c1857 (nin5)
T I
1. Ligate the insert DNA

encoding the target protein(s)
into the HybriZAP-2.1 vector
3. Ligate insert DNA encoding 2. Convert the HybriZAP-2.1
the bait protein into the pBD- library to the pAD-GAL4-2.1
GAL4 Cam phagemid vector library by mass excision
P ADH1 P ADH1
2-micron ori GAL4-BD 2-micron ori GAL4-AD
Target DNA insert
Bait DNA Insert T ADH1
T ADH1
pBD-GAL4 Cam pAD-GAL4-2.1
ampicillin
chloramphenicol LEU2
TRP1
pUC ori
pUC ori f1 ori
f1 ori
4. Coexpress the target and bait proteins in yeast

5. Assay for transcriptional activation of reporter genes
6. Verify interaction by secondary screening
FIGURE 1 The HybriZAP-2.1 two-hybrid vector system. DNA inserts are ligated into the HybriZAP-2.1 vector to generate the
primary lambda library. This primary lambda library is amplified and converted by in vivo mass excision to a pAD-GAL4-2.1
library. DNA that expresses a library of the GAL4 AD hybrid proteins (target proteins or Y) is isolated from E. coli. DNA
encoding the bait protein is inserted into the pBD-GAL4 Cam phagemid vector for expression of the GAL4 BD hybrid protein
(bait protein or X). The bait and target plasmids are transformed and coexpressed in the yeast host, YRG-2 strain. Colonies
that contain DNA encoding target proteins, which interact with the bait protein, are identified by transcription of the HIS3 and
lacZ reporter genes in the yeast host strain.
The pAD-GAL4-2.1 phagemid vector contains the ampicillin-resistance gene [β-lactamase (bla)] for selection with ampicillin
in E. coli. The pBD-GAL4 Cam phagemid vector contains the chloramphenicol-resistance gene [chloramphenicol
acetyltransferase] and promoter for selection with chloramphenicol in E. coli. For selection in yeast, the pAD-GAL4-2.1
phagemid vector contains the LEU2 gene and the pBD-GAL4 Cam phagemid vector contains the TRP1 gene. Hybrid proteins
are expressed in yeast from the ADH1 promoter (P ADH1) and terminated by the ADH1 terminator (T ADH1).

FIGURE 2 Detection of interacting proteins by transcription of the lacZ reporter gene. The GAL4 UAS and the lacZ
reporter gene are integrated into the yeast chromosome. (A) The GAL4 BD hybrid protein (BD and the bait protein X)
binds to the GAL4 UAS present upstream of the lacZ reporter gene. The GAL4 AD hybrid protein (AD and the target
protein Y) binds transcription factors in the nucleus but does not localize to the GAL4 UAS. (B) If the bait (X) and target
(Y) proteins interact, the GAL4 AD and the GAL4 BD are brought close to each other and act together with the bound
transcription factors to initiate transcription of the lacZ reporter gene.
VECTORS
The HybriZAP-2.1 vector will accommodate DNA inserts from 0 to 6 kb in
length. In vivo mass excision allows conversion of the HybriZAP-2.1
lambda library to a pAD-GAL4-2.1 phagemid library by the same excision
mechanism found in the Lambda ZAP® vectors.4, 5, 6
The HybriZAP-2.1 lambda vector and the pAD-GAL4-2.1 phagemid vector

contain a multiple cloning site (MCS) with BamH I, Nhe I, EcoR I, Xho I,
Sal I, Xba I, Pst I, and Bgl II restriction sites. The pBD-GAL-4 Cam
phagemid vector contains an MCS with EcoR I, Srf I, Sma I, Xho I, Sal I,
Xba I, and Pst I restriction sites (Figures 3–5 and Table I). The unique
EcoR I and Xho I cloning sites in the HybriZAP-2.1 lambda vector and the
pAD-GAL4-2.1 vector make these vectors compatible with Stratagene’s
cDNA Synthesis Kit for the preparation of unidirectional cDNA libraries.
The unique EcoR I and Sal I cloning sites are used for the preparation of
cDNA libraries in the pBD-GAL4 Cam phagemid vector because the Xho I
site in the MCS is not unique. The unique BamH I, Nhe I, and EcoR I sites
at the 5´ end and the Xho I, Sal I, Xba I, and Bgl II sites at the 3´ end of the
DNA insert facilitate the transfer of DNA encoding the target protein into
commonly used protein expression/purification vectors. The Xba I site in the
HybriZAP-2.1 lambda vector and pAD-GAL4-2.1 and pBD-GAL4 Cam
phagemid vectors contains the UAG amber suppressor in the same
translational reading frame as the GAL4 domain. DNA should therefore be
inserted such that the Xba I site is not between the GAL4 domain and the
DNA insert. In the HybriZAP-2.1 lambda vector and pBD-GAL4 Cam
phagemid vector, the Xba I site is not unique.

The pAD-GAL4-2.1 and pBD-GAL4 Cam phagemid vectors contain the
pUC origin for replication and an f1 origin for production of single-stranded
DNA (ssDNA) in E. coli. Single-stranded DNA can be used for DNA
sequencing or site-directed mutagenesis. The pAD-GAL4-2.1 and
pBD-GAL4 Cam phagemid vectors contain ampicillin-resistance gene
[β-lactamase (bla)] and chloramphenicol acetyltransferase genes,
respectively, for selection with ampicillin and chloramphenicol in E. coli.
The pAD-GAL4-2.1 and pBD-GAL4 Cam phagemid vectors contain the
2μ origin for replication in yeast cells. For selection in yeast, the
pAD-GAL4-2.1 phagemid vector contains the LEU2 gene and the
pBD-GAL4 Cam phagemid vector contains the TRP1 gene. In both vectors,
the hybrid protein is expressed by the alcohol dehydrogenase 1 (ADH1)
promoter (P ADH1) and is terminated by the ADH1 terminator (T ADH1).
HybriZAP®-2.1 Vector Map
Right End 45.52 kb

BamH I 24.34
Hind III 27.17
Hind III 36.59

Hind III 37.16
Hind III 41.15

Left End 0 kb
Nae I 19.52
Bgl II 24.44
Not I 26.84
Pvu I 11.93
Mlu I 0.46
Sfi I 20.08
Mlu I 5.5
A-J att int xis c1857 (nin5)

T I
MCS
pAD-GAL4-2.1
FIGURE 3 Restriction map of the HybriZAP-2.1 vector. The HybriZAP-2.1 vector contains
lambda genes A through J in the left arm and att, int, xis, and cI857 in the right arm. The
f1 initiator (I) and terminator (T) allow efficient in vivo excision of the pAD-GAL4-2.1
phagemid vector from the HybriZAP-2.1 vector.

TABLE I
Unique Restriction Sites in the MCS
Restriction site HybriZAP®-2.1 pAD-GAL4-2.1 pBD-GAL4 Cam
in MCS vector phagemid vector phagemid vector
BamH I No Yes No site
Nhe I No Yes No site
EcoR I Yes Yes Yes
Xho I Yes Yes No
Sal I No Yes Yes
Xba I a
No Yes No
Pst I No Yes Yes
Srf I No site No site Yes
Sma I No site No site No
Bgl II No Yes No site
a
The Xba I site in the HybriZAP-2.1 lambda vector and pAD-GAL4-2.1 and
pBD-GAL4 Cam phagemid vectors contains the UAG amber suppressor in the same
translational reading frame as the GAL4 domain. DNA should therefore be inserted
such that the Xba I site is not between the GAL4 domain and the DNA insert.
Library Construction in the HybriZAP®-2.1 Vector

The library was synthesized using the ZAP-cDNA® synthesis method.6 For
unidirectional libraries, the linker–primer was designed with a GAGA
sequence to protect the Xho I restriction enzyme recognition site and an
18-base poly(dT) sequence. The restriction site allows the finished cDNA to
be inserted into the vector unidirectionally in the sense orientation. The
linker–primer is a 50-base oligonucleotide with the following sequence:
5´-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT-3´
"GAGA" Sequence Xho I Poly(dT)
The adapters are comprised of 9- and 13-mer oligonucleotides, which are

complementary to each other and have an EcoR I cohesive end. The
adapters have the following sequence:
5´-OH-AATTCGGCACGAG-3´
3´-GCCGTGCTCp-5´

pAD-GAL4-2.1 Vector Map
P ADH1
2-micron ori GAL4-AD
MCS
T ADH1
pAD-GAL4-2.1
ampicillin 7.7 kb
LEU2
pUC ori
f1 ori
pAD-GAL4-2.1 Multiple Cloning Site Region

(sequence shown 812–958)
end of GAL4 activation domain BamH I Nhe I EcoR I
5´ CCA AAC CCA AAA AAA GAG ATC GAA TTA GGA TCC TCT GCT AGC AGA GAA TTC AAT...
Xho I Sal I Xba I

T7 promoter
...TCT CTA ATG CTT CTC GAG AGT ATT AGT CGA CTC TAG AGC CCT ATA GTG AGT CGT ATT...
Pst I Bgl II
...ACT GCA GAG ATC TAT GAA TCG TAG ATA CTG AAA AAC 3´
STOP STOP STOP
Feature Nucleotide position

yeast ADH1 promoter 4–408
GAL4 activation domain (114 amino acids) 488–829
multiple cloning site 839–935
yeast ADH1 terminator 1168–1318
yeast LEU2 selection marker ORF 1615–2709
f1 origin of ssDNA replication 3483–3789
pUC origin of replication 4427–5094
ampicillin resistance (bla) ORF 5245–6102
2μ yeast origin of replication 6489–7653
FIGURE 4 Circular map features of the excised pAD-GAL4-2.1 phagemid vector. The Xba I site contains the UAG amber
suppressor in the same translational reading frame as the GAL4 domain. DNA should therefore be inserted such that the
Xba I site is not between the GAL4 domain and the DNA insert. The complete sequence and list of restriction sites can be
found at www.stratagene.com.

The pBD-GAL4 Cam Vector Map P ADH1
2-micron ori GAL4-BD
MCS
T ADH1
TRP1
pBD-GAL4 Cam
6.5 kb
chloramphenicol
f1 ori
pUC ori
pBD-GAL4 Cam Multiple Cloning Site Region

(sequence shown 854–992)
end of GAL4 binding domain EcoR I Srf I Sal I
5´ CAA AGA CAG TTG ACT GTA TCG CCG GAA TTC GCC CGG GCC TCG AGC CCG GGT CGA...
T7 promoter
...CTC TAG AGC CCT ATA GTG AGT CGT ATT ACT GCA GCC AAG CTA ATT CCG GGC GAA...
...TTT CTT ATG ATT TAT GAT TTT TAT TAT TAA A 3´
STOP STOP STOP
Feature Nucleotide position

yeast ADH1 promoter 4–408
GAL4 DNA-binding domain (148 amino acids) 434–877
multiple cloning site 878–941
yeast ADH1 terminator 948–1154
yeast TRP1 selection marker ORF 1197–1871
f1 origin of ssDNA replication 2322–2628
pUC origin of replication 2970–3637
chloramphenicol resistance ORF 4174–4725
2μ yeast origin of replication 5330–6489
FIGURE 5 Circular map and features of the pBD-GAL4 Cam phagemid vector. In the MCS of the pBD-GAL4 Cam phagemid
vector, the EcoR I, Srf I, Sal I, and Pst I sites are unique; however, the Xho I, Sma I and Xba I sites are not. The Xba I site
contains the UAG amber suppressor in the same translational reading frame as the GAL4 domain. DNA should therefore be
inserted such that the Xba I site is not between the GAL4 domain and the DNA insert. The complete sequence and list of
restriction sites can be found at www.stratagene.com.

BACTERIAL HOST STRAINS
The table in the appendix compares the qualities and features of E. coli and
yeast host strains (see Appendix: General Comparison of Escherichia coli
versus Yeast Host Strains).
Bacterial Strain Genotypes

XL1-Blue MRF´ Strain Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1
supE44 thi-1 recA1 gyrA96 relA1 lac [F´ proAB lacIqZΔM15 Tn10 (Tetr)]
XLOLR Strain Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 thi-1 recA1

gyrA96 relA1 lac [F´ proAB lacIqZΔM15 Tn10 (Tetr)] Su– (nonsuppressing)
λR (lambda resistant)
Note Use the XLOLR strain for plating excised phagemids and the
XL1-Blue MRF´ strain for all other manipulations.
XL1-Blue MRF´ Bacterial Strain Description

The RecA– E. coli host strain XL1-Blue MRF´ is supplied with the
HybriZAP-2.1 two-hybrid libraries.7 The episome is selectively maintained
by the presence of the Tn10 tetracycline-resistance gene on the F´ episome
in the XL1-Blue MRF´ strain. It is the ideal strain for amplification and
excisions.
Note The mcrA, mcrCB, and mrr mutations prevent restriction of

methylated DNA, making XL1-Blue MRF´ compatible with cloning
cDNA constructed using Stratagene's cDNA Synthesis Kit.
The strains used for the Lambda gt11 vector (i.e., Y1088, Y1089, and
Y1090) are not suitable for use with the HybriZAP-2.1 vector because these
strains contain the plasmid pMC9, a pBR322 derivative, which contains
many of the same sequences as those found in the phagemid portion of the
HybriZAP-2.1 vector.
The F´ episome present in the XL1-Blue MRF´ strain contains the genes for
expression of the bacterial F´ pili required for filamentous (i.e., f1 or M13)
phage infection. The conversion of the HybriZAP-2.1 vector to the
pAD-GAL4-2.1 phagemid vector requires superinfection with a filamentous
helper phage. (This efficient in vivo excision process is outlined in In Vivo
Excision of the pAD-GAL4-2.1 Phagemid Vector from the HybriZAP-2.1
Vector.)

Recommended Media
Agar plates and Agar plates
liquid medium for Liquid medium for and top agar Agar plates
bacterial streak bacterial cultures prior for plaque for excision
Host strain and glycerol stock to phage attachment formation protocol
XL1-Blue MRF´ strain LB-tetracyclinea LB broth with supplementsa-c NZYa —
XLOLR strain LB-tetracycline a
LB broth with supplements a-c
— LB-ampicillina
a
See Preparation of Media and Reagents.
b
LB with 0.2% (w/v) maltose and 10 mM MgSO4.
c
Maltose and magnesium supplements are required for optimal lambda phage receptor expression on the surface of
the XL1-Blue MRF’ host cell. These media supplements are not required for helper phage infection, but are included
in both protocols for simplified media preparation.
Establishing an Agar Plate Bacterial Stock

The bacterial host strains have been sent as bacterial glycerol stocks. On
arrival, prepare the following from the bacterial glycerol stock using the
appropriate media as indicated in the previous table:
Note The host strains may thaw during shipment. The vials should be
stored immediately at –20° or –80°C, but most strains remain
viable longer if stored at –80°C. It is also best to avoid repeated
thawing of the host strains in order to maintain
extended viability.
1. Revive the stored cells by scraping off splinters of solid ice with a
sterile wire loop.
2. Streak the splinters onto an LB agar plate containing the appropriate

antibiotic, if one is necessary.
3. Incubate the plate overnight at 37°C.
4. Seal the plate with Parafilm® laboratory film and store the plate at 4°C
for up to 1 week.
5. Restreak the colonies onto a fresh plate every week.
Preparation of a –80°C Bacterial Glycerol Stock

1. In a sterile 50-ml conical tube, inoculate 10 ml of appropriate liquid
medium containing antibiotic with one or two colonies from the plate.
Grow the cells to late log phase (OD600 = 0.8–1.0).
2. Add 4.5 ml of a sterile glycerol–liquid medium solution (prepared by

mixing 5 ml of glycerol + 5 ml of appropriate medium) to the bacterial
culture from step 1. Mix well.
3. Aliquot into sterile centrifuge tubes (1 ml/tube).
This preparation may be stored at –20°C for 1–2 years or at –80°C for more
than 2 years.

Library Titer Determination
Preparation of the Plating Culture
Note XL1-Blue MRF´ cells are RecA– and consequently grow slowly. .
1. Streak the XL1-Blue MRF´ bacterial glycerol stock onto

LB-tetracycline agar plates. Incubate the plates overnight at 37°C.
2. Inoculate 50 ml of LB broth with supplements in a sterile flask with a

single XL1-Blue MRF´ colony.
3. Grow at 37°C with shaking for 4–6 hours. Do not grow past an OD600
of 1.0. Alternatively, grow overnight at 30°C with shaking at 200 rpm.
(The lower temperature keeps the bacteria from overgrowing, which
reduces the number of nonviable cells. Phage adherence to nonviable
cells results in a decreased titer.)
4. Spin the cells at 500 × g for 10 minutes and discard the supernatant.
5. Gently resuspend the cells in half the original volume with sterile
10 mM MgSO4. (Do not vortex.)
Note For later use, store the cells at 4°C overnight in

10 mM MgSO4.
6. Dilute the cells to an OD600 of 0.5 with sterile 10 mM MgSO4.
Note The bacteria should be used immediately following dilution.
Measuring the Titer

Notes The XL1-Blue MRF´ strain is RecA– McrA– and McrCB– Mrr–
and does not restrict methylated DNA. Use of any other cell
line may result in dramatically reduced titer.
In order to obtain accurate titers, use freshly prepared
XL1-Blue MRF´ cells.
7. To determine the titer of the library, dilute the amplified phage stock in
SM buffer by the following amounts: 1:10,000, 1:100,000, 1:1,000,000.

8. Mix the following components:
1 μl of the phage library at the appropriate dilution

200 μl of XL1-Blue MRF´ cells at an OD600 of 0.5 (from step 6 above)
Incubate the mixture at 37°C for 15 minutes to allow the phage to

attach to the cells. (Best results are obtained with gentle shaking.)
9. Add 2–3 ml of NZY top agar held at 48°C (see Preparation of Media
and Reagents).
10. Plate immediately onto NZY agar plates and allow the plates to set
undisturbed for 10 minutes.
11. Place the plates upside-down in a 37°C incubator. Plaques should be

visible after 6–8 hours.

YEAST HOST STRAIN
TABLE II
Yeast Host Strain
Reporter Transformation
Strain Genotype genes markers
YRG-2a MATa ura3-52 his3-200 ade2-101 lys2- lacZ, HIS3 leu2, trp1
801 trp1-901 leu2-3 112 gal4-542
gal80-538 LYS2::UASGAL1-TATA GAL1-HIS3
URA3::UASGAL4 17mers(x3)-TATACYC1-lacZ
a
The LYS2 gene in this strain is nonfunctional.
Yeast Strain Genotype and Phenotypic Verification

Table II gives the genotype of the YRG-2 yeast host strain.
The phenotype of the yeast host strain should be verified as outlined below
prior to performing the HybriZAP-2.1 two-hybrid vector system assays.
1. Prepare a fresh plate of the yeast host strain on a YPAD agar plate (see
Preparation of Media and Reagents) from the yeast glycerol stock as
outlined below:
a. Revive the stored cells by scraping off splinters of solid ice with a
sterile wire loop.
b. Streak the splinters onto a YPAD agar plate.
c. Incubate the plate at 30°C for 2–3 days.
2. Prepare SD agar plates using the appropriate 10× dropout solution (see
Synthetic Minimal Medium in the Two-Hybrid Vector System Media
and Reagents subsection of Preparation of Media and Reagents) to test
the yeast host strain for the following nutritional requirements:
tryptophan (Trp), leucine (Leu), histidine (His), and uracil (Ura). Streak
the yeast host strain onto the agar plates containing the appropriate
10× dropout solution and incubate the plates at 30°C for 2–3 days.
The yeast host strain should grow only on the SD agar plates without
Ura. The yeast host strain may grow slightly on the SD agar plates
without His due to leaky expression of the HIS3 gene. The yeast host
strain should not grow on the SD agar plates without Trp or Leu.
Although the pGAL1, which governs expression of the HIS3 gene, is
slightly leaky, the addition of the histidine antimetabolite,
3-aminotriazole, to restore histidine auxotrophy is not necessary.
3-Aminotriazole slows the growth rate of the yeast cells and has not
been shown to be effective at reducing background growth.
3. After the phenotype has been verified, use the tested colony to
inoculate medium for the preparation of competent yeast cells.

Yeast Strain Description
The HybriZAP-2.1 two-hybrid cDNA libraries kit includes the YRG-2
strain, a yeast strain with two reporter genes (see Table II) for the detection
of protein–protein interactions in vivo. The YRG-2 strain is a derivative of
the HF7c strain8 and was selected for its ability to generate high-efficiency
competent cells.9 The YRG-2 strain carries a mutation which ensures that
the endogenous GAL4 gene is not expressed. In addition, GAL80, whose
product inhibits function of the GAL4 gene product, is mutated. The YRG-2
strain carries the auxotrophic markers leucine (leu2) and tryptophan (trp1),
for selection of yeast which have been transformed with the AD or BD
plasmids, respectively. YRG-2 also carries the auxotrophic marker histidine
(his3), for selection of yeast which have been transformed with plasmids
encoding interacting proteins that together activate transcription from the
HIS3 reporter. For generalized protocols and techniques used to analyze the
10
genetics and molecular biology of yeast, see Reference 10.
The YRG-2 strain contains a dual two-hybrid assay system with lacZ and
HIS3 reporter gene constructs. The lacZ reporter gene construct consists of
three copies of the GAL4 17-mer consensus sequence (GAL4 DNA-binding
sites) and the TATA portion of the iso-1-cytochrome c (CYC1) promoter
(pCYC1), which are fused to the lacZ reporter gene and regulate its
expression. The lacZ reporter gene construct, including the LYS2 yeast
gene,* has been integrated into the nonfunctional lys locus. The HIS3
reporter gene construct consists of the UASGAL1, which contains four GAL4
DNA-binding sites, and the TATA portion of the GAL1 promoter (pGAL1),
which are fused to the HIS3 reporter gene and regulate its expression. The
HIS3 reporter gene construct, including the URA3 yeast gene, has been
integrated into the nonfunctional ura locus. Expression of the functional
URA3 yeast gene allows the YRG-2 strain to grow in the absence of uracil.
The GAL4 BD hybrid protein binds to the UASGAL1 and the GAL4 17-mers
present upstream of the reporter genes. If X and Y proteins interact, the AD
and the BD are brought in close proximity to each other and act together to
initiate transcription of the reporter genes (see Figure 2B).
* The LYS2 gene in this strain is nonfunctional.

Preparation of the Yeast Host Strain
The yeast host strain has been sent as a yeast glycerol stock. On arrival,
prepare the following from the yeast glycerol stock. For the appropriate
medium, please refer to the following table:
Host strain Agar plate for yeast streak Medium for yeast glycerol stock
YRG-2 strain YPADa,b
YPADa,b
a
b
Adenine sulfate is added to the medium to reduce the reversion rate of the ade2-101
mutation, thereby reducing the amount of reddish pigment in the yeast colonies.
Note The yeast host strain should be stored immediately at –80°C. It is

also best to avoid repeated thawing of the yeast host strain in
order to maintain extended viability.
1. Revive the stored cells by scraping off splinters of solid ice with a
sterile wire loop.
2. Streak the splinters onto a YPAD agar plate.
3. Incubate the plate at 30°C until colonies appear (~2–3 days).
4. Seal the plate with Parafilm laboratory film and store the plate at 4°C
for up to 1 week.
5. Restreak the colonies onto a fresh plate every week.
Preparation of a –80°C Yeast Glycerol Stock

1. In a sterile 50-ml conical tube, inoculate 10 ml of YPAD broth with
one colony from the plate. Grow the cells to late log phase
(OD600 = 0.8–1.0).
2. Add 4.5 ml of a sterile glycerol–liquid medium solution (prepared by

mixing 5 ml of glycerol + 5 ml of appropriate medium) to the yeast
culture from step 1. Mix well.
3. Aliquot into sterile centrifuge tubes (1 ml/ tube).
This preparation may be stored at –80°C for more than 2 years.

HELPER PHAGE
The ExAssist interference-resistant helper phage with XLOLR strain is
designed to efficiently excise the pAD-GAL4-2.1 phagemid vector from the
HybriZAP-2.1 vector while preventing problems associated with helper
phage co-infection. The ExAssist helper phage contains an amber mutation
that prevents replication of the phage genome in a nonsuppressing E. coli
strain (e.g., XLOLR cells). Only the excised phagemid can replicate in the
host, removing the possibility of co-infection from the ExAssist helper
phage. Because ExAssist helper phage cannot replicate in the XLOLR
strain, single-stranded rescue cannot be performed in this strain using
ExAssist helper phage. XLOLR cells are also resistant to lambda infection,
thereby ensuring that the library is not lysed by residual lambda phage.
Note The ExAssist helper phage is recommended only for excision of the
pAD-GAL4-2.1 phagemid vector from the HybriZAP-2.1 vector. It
should not be used for single-stranded rescue in general, because
this f1 helper phage possesses α-complementing
β-galactosidase sequences which may interfere with sequencing or
site-directed mutagenesis where oligonucleotide primers hybridize
to β-galactosidase sequences [e.g., M13 (–20) primer].
Storing the Helper Phage

The ExAssist helper phage is supplied in 7% dimethylsulfoxide (DMSO)
and should be stored at –80°C. If the titer drops over time, prepare a fresh
high-titer stock of the helper phage as outlined in Amplifying the Helper
Phage. It is important to titer the ExAssist helper phage prior to each use.
Expect titers of approximately 1010 pfu/ml.
Titering the Helper Phage

Titer the helper phage with XL1-Blue MRF´ cells:
1. Transfer a colony of XL1-Blue MRF´ cells into 10 ml of LB broth with

supplements in a 50-ml conical tube. Incubate the conical tube with
shaking at 37°C until growth reaches an OD600 of 1.0.
2. Dilute the phage (10–4–10–7) in SM buffer§ and combine 1 μl of each

dilution with 200 μl of the XL1-Blue MRF´ cells (OD600 = 1.0).
3. Incubate the helper phage and the XL1-Blue MRF´ cells for 15 minutes
at 37°C to allow the phage to attach to the cells.
4. Add 3 ml of NZY top agar, melted and cooled to ~48°C, and then pour
immediately onto prewarmed NZY agar plates.
Note ExAssist plaques will have a cloudier appearance than
lambda phage plaques.
§

5. Incubate the plates overnight at 37°C.
6. To determine the titer [in plaque-forming units per milliliter (pfu/ml)],

use the following formula:
⎡ Number of plaques ( pfu) × dilution factor ⎤

⎢ ⎥ × 1000 μl / ml
⎣ Volume plated ( μl ) ⎦
where the volume plated (in microliters) refers to the volume of the
helper phage solution added to the cells.
Amplifying the Helper Phage
1. Transfer a colony of XL1-Blue MRF´ cells from a fresh

LB-tetracycline plate into 10 ml of LB broth with supplements in a
50-ml conical tube.
2. Incubate the conical tube with shaking at 37°C until growth reaches an
OD600 of 0.3.
Note An OD600 of 0.3 corresponds to 2.5 × 108 cells/ml.
3. Add the helper phage at a multiplicity of infection (MOI) of 20:1

(phage-to-cells ratio).
4. Incubate the conical tube at 37°C for 15 minutes to allow the phage to
attach to the cells.
5. Incubate the conical tube with shaking at 37°C for 8 hours.
6. Heat the conical tube at 65°C for 15 minutes.
7. Spin down the cell debris and transfer the supernatant to a fresh
conical tube.
8. The titer of the supernatant should be between 7.5 × 1010 and

1.0 × 1012 pfu/ml for ExAssist helper phage.
9. Add dimethylsulfoxide (DMSO) to a final concentration of 7% (v/v)

and store at –80°C.
For a helper phage titering protocol, please see Titering the Helper Phage.

CONTROL PLASMIDS
Description
The HybriZAP-2.1 two-hybrid vector system contains six control plasmids
(see Table III and Figure 6). The pGAL4 control plasmid expresses the
entire coding sequence of the wild-type GAL4 protein.2 The pBD-WT
control plasmid expresses the DNA-binding domain (BD) of GAL4 and
amino acids (aa) 132–236 of wild-type lambda cI, fragment C, as a hybrid
protein.11, 12 The pAD-WT control plasmid expresses the activation domain
(AD) of GAL4 and aa 132–236 of wild-type lambda cI, fragment C, as a
hybrid protein. The pAD-MUT control plasmid expresses the AD of GAL4
and aa 132–236 of E233K mutant lambda cI, fragment C, as a hybrid
protein.13, 14 The pBD-MUT control plasmid expresses the BD of GAL4 and
aa 132–236 of E233K mutant lambda cI, fragment C, as a hybrid protein.
The lambda cI gene product (cI-wt) naturally forms homodimers. The
cI-E233K mutation encodes a substitution in the gene product that interferes
with the interaction between the homodimers, resulting in a weaker protein–
protein interaction. The pLamin C control plasmid expresses the BD of
GAL4 and aa 67–230 of human lamin C as a hybrid protein.15
Applications
These plasmids are used alone or in pairwise combination as positive and
negative controls for the induction of the HIS3 and lacZ genes (Tables IV
and V). Induction of the HIS3 gene enables the transformed host to grow on
SD medium without His. Induction of the lacZ gene is detected by cleavage
of a chromogenic substrate causing the transformed host to turn blue in
color. The pGAL4 control plasmid can be used alone to verify that induction
of the lacZ and HIS3 genes has occurred and that the gene products are
detectable in the assay used. The pLamin C control plasmid can be used in
pairwise combination with the pAD-WT control plasmid and/or the
pAD-MUT control plasmid to verify that the lacZ and HIS3 genes are not
induced, as the proteins expressed by each of these pairs do not interact
in vivo.
Two pair of control plasmids are used as positive controls to verify that
induction of the HIS3 and lacZ genes has occurred. The degree of color
development of the transformed host depends on the strength of interaction
of the expressed proteins. The pBD-WT and pAD-WT control plasmids
express proteins that interact strongly (Kd = 20 nM) in vivo, and the
transformed host turns blue in color. The pBD-MUT and pAD-MUT control
plasmids express proteins that interact weakly (Kd = 200 nM) in vivo, and
the transformed host turns light blue in color.

TABLE III
Description of the Control Plasmids
Control Insert
plasmid descriptiona Vector Genotype Function
r
pGAL4 Wild-type, full- pRS415 LEU2, Amp Positive control
length GAL4
pBD-WT Wild-type fragment pBD-GAL4 Cam TRP1, Camr Interaction
C of lambda cI control
repressor
(aa 132–236)
pAD-WT Wild-type fragment pAD-GAL4-2.1 LEU2, Ampr Interaction
C of lambda cI control
repressor
(aa 132–236)
pBD-MUT E233K mutant pBD-GAL4 Cam TRP1, Camr Interaction
fragment of lambda control
cI repressor
(aa 132–236)
pAD-MUT E233K mutant pAD-GAL4-2.1 LEU2, Ampr Interaction
fragment of lambda control
cI repressor
(aa 132–236)
pLamin C Human lamin C pBD-GAL4 TRP1, Ampr Negative
(aa 67–230) control
a
aa, Amino acid.

2-micron ori LEU2
f1 ori
ampicillin
pUC ori
pGAL4
11.5 kb
GAL4 (full-length)
P ADH1 P ADH1
2-micron ori GAL4-BD 2-micron ori GAL4-BD
cI-wt (aa 132-236) cI-E233K (aa 132-236)
T ADH1 T ADH1
pBD-WT pBD-MUT
6.8 kb 6.8 kb
TRP1 TRP1
chloramphenicol chloramphenicol
f1 ori f1 ori
pUC ori pUC ori
P ADH1 P ADH1
2-micron ori GAL4-AD 2-micron ori GAL4-AD
cI-wt (aa 132-236) cI-E233K (aa 132-236)
T ADH1 T ADH1
ampicillin pAD-WT ampicillin pAD-MUT

8.0 kb 8.0 kb
LEU2 LEU2
pUC ori pUC ori
f1 ori f1 ori
P ADH1
2-micron ori GAL4-BD
lamin C (aa 67-230)

T ADH1
pLamin C
7.0 kb TRP1
ampicillin
f1 ori
pUC ori
FIGURE 6 Circular maps of the control plasmids.

Expected Results for Control Plasmid Assays
The expected results for transformation of the control plasmids alone or in
pairwise combination into the YRG-2 strain when plated on selective media
and assayed for expression of the lacZ gene are outlined in Tables IV and V.
TABLE IV
Expected Results for the pGAL4 Positive Controla
Control plasmid Expected results
pGAL4 Growth, blue
a
When transformed into the YRG-2 competent cells, plated on SD agar plates without
Leu and assayed for expression of the lacZ reporter gene.
TABLE V
Expected Results for Interaction Control Plasmidsa
Control plasmids Expected results
SD agar SD agar
SD agar SD agar plates plates
plates plates without without
BD AD without without Leu and Leu, Trp,
fusion fusion Leu Trp Trp and His
pBD-WT Growth,
white
pBD-MUT Growth,
white
pAD-WT Growth,
white
pAD-MUT Growth,
white
pLamin C Growth,
white
pBD-WT pAD-WT Growth, Growth,
blue blue
pBD-MUT pAD-MUT Growth, Growth,
light blue light blue
pLamin C pAD-WT Growth, No growth
white
pLamin C pAD-MUT Growth, No growth
white
a
When transformed into YRG-2 competent cells, plated on the SD media indicated, and
assayed for expression of the lacZ reporter gene.

IN VIVO EXCISION OF THE PAD-GAL4-2.1 PHAGEMID VECTOR
FROM THE HYBRIZAP®-2.1 VECTOR
Converting the HybriZAP-2.1 two-hybrid library to the phagemid form
allows screening of the phagemid library in yeast cells by transformation of
yeast cells with supercoiled phagemid DNA. The HybriZAP-2.1 vector has
been designed to allow simple, efficient in vivo excision of any cloned insert
contained within the lambda vector to form a phagemid containing the
cloned insert. 4, 5, 6
This in vivo excision depends on the DNA sequences that Stratagene has
placed in the HybriZAP-2.1 vector and on the presence of a variety of
proteins, including helper phage-derived proteins. The helper phage proteins
recognize a region of DNA normally serving as the f1 bacteriophage "origin
of replication" for positive-strand synthesis. This origin of replication can be
divided into two overlying parts: (1) the site of initiation and (2) the site of
termination for DNA synthesis.16 These two regions have been subcloned
separately into the HybriZAP-2.1 vector. The lambda phage is made
accessible to the helper phage-derived proteins by simultaneously infecting
a strain of E. coli with both the lambda vector and the helper phage.
Inside E. coli, the helper phage-derived proteins recognize the initiator DNA
that is within the lambda vector. One of these proteins then nicks one of the
two DNA strands. At the site of this nick, new DNA synthesis begins and
duplicates whatever DNA exists in the lambda vector "downstream" (3´) of
the nicking site. DNA synthesis of a new single strand of DNA continues
through the cloned insert until a termination signal, positioned 3´ of the
initiator signal, is encountered within the constructed lambda vector. The
ssDNA molecule is circularized by the gene II product from the helper
phage, forming a circular DNA molecule containing the DNA between the
initiator and terminator. In the case of the HybriZAP-2.1 vector, this
includes all sequences of the pAD-GAL4-2.1 phagemid vector and the
insert, if one is present. This conversion is the "subcloning" step, since all
sequences associated with normal lambda vectors are positioned outside of
the initiator and terminator signals and are not contained within the
circularized DNA. In addition, the circularizing of the DNA automatically
recreates a functional f1 origin as found in f1 bacteriophage or phagemids.
Signals for "packaging" the newly created phagemid are linked to the f1
origin sequence. The signals permit the circularized ssDNA to be
"packaged" into phagemid particles and secreted from the E. coli. Following
secretion of the phagemid particle, the E. coli cells used for in vivo excision
of the cloned DNA are killed and the lambda phage is lysed by heat
treatment at 70°C. The phagemid is not affected by the heat treatment.
Escherichia coli is infected with the phagemid and can be plated on
selective media to form colonies. DNA from colonies can be used for
analysis of insert DNA, including DNA sequencing, subcloning, and
mapping. Colonies from the excised pAD-GAL4-2.1 phagemid vector can
also be used for subsequent production of ssDNA suitable for dideoxy-
sequencing and site-specific mutagenesis.

ExAssist® Helper Phage and XLOLR Strain
The ExAssist helper phage, used with the XLOLR strain, is designed to
efficiently excise the pAD-GAL4-2.1 phagemid vector from the
HybriZAP-2.1 vector, while eliminating problems associated with helper
phage co-infection. The ExAssist helper phage contains an amber mutation
that prevents replication of the helper phage genome in a nonsuppressing
E. coli strain such as XLOLR cells. This allows only the excised phagemid
to replicate in the host, removing the possibility of productive co-infection
from the ExAssist helper phage. Since the ExAssist helper phage cannot
replicate in the XLOLR strain, single-stranded rescue cannot be performed
in this strain using this helper phage.
Mass Excision Protocol

Note The ratios of bacterial cells, HybriZAP-2.1 library phage particles
and ExAssist helper phage strongly influence excision efficiency.
For a library titering protocol, see Library Titer Determination.
If the ExAssist helper phage has been stored at 4°C for >1 month
or passed through a freeze–thaw cycle, titer the helper phage with
XL1-Blue MRF´ cells prior to use (see Titering the Helper Phage).
Day 1
1. Grow separate 50-ml overnight cultures of XL1-Blue MRF´ and

XLOLR cells in LB broth with supplements at 30°C.
Day 2
2. Gently spin down the XL1-Blue MRF´ and XLOLR cells (1000 × g).
Resuspend each of the cell pellets in 25 ml of 10 mM MgSO4. Adjust
the cell concentration to an OD600 of 1.0 (8 × 108 cells/ml) in
10 mM MgSO4.

3. In a 50-ml conical tube, combine a portion of the amplified lambda
bacteriophage library with XL1-Blue MRF´ cells at a MOI of 1:10
lambda phage-to-cell ratio. Excise 10- to 100-fold more lambda phage
than the size of the primary library to ensure statistical representation of
the excised clones. Add ExAssist helper phage at a 10:1 helper phage-
to-cells ratio to ensure that every cell is co-infected with lambda phage
and helper phage.
For example, use

107 pfu of the lambda phage (i.e., 10- to 100-fold above the
primary library size)
108 XL1-Blue MRF´ cells (1:10 lambda phage-to-cell ratio, noting
that an OD600 of 1.0 corresponds to 8 × 108 cells/ml)
109 pfu of ExAssist helper phage (10:1 helper phage-to-cells ratio)
Note Briefly spin the lambda phage stock to ensure that the
chloroform is separated completely before removing the
aliquot used in the excision reaction.
4. Incubate the conical tube at 37°C for 15 minutes to allow the phage to
attach to the cells.
5. Add 20 ml of LB broth with supplements and incubate the conical tube

for 2.5–3 hours at 37°C with shaking.
Notes Incubation times for mass excision in excess of 3 hours may
alter the clonal representation.
The turbidity of the media is not indicative of the success of

the excision.
6. Heat the conical tube at 65–70°C for 20 minutes to lyse the lambda
phage particles and the cells. Spin down the debris at 1000 × g for
10 minutes.
7. Transfer the supernatant into a fresh sterile conical tube. This stock
contains the excised pAD-GAL4-2.1 phagemid packaged as
filamentous phage particles. (This stock may be stored at 4°C for
1–2 months.)
8. To titer the excised phagemids, combine 1 μl of this supernatant with

200 μl of the XLOLR cells from step 2 in a 1.5-ml microcentrifuge
tube.
9. Incubate the microcentrifuge tube at 37°C for 15 minutes.
10. Plate 100 μl of the cell mixture onto LB–ampicillin (100 μg/ml) agar
plates and incubate the plates overnight at 37°C.
Note It may be necessary to further dilute the cell mixture to

achieve single-colony isolation.

Day 3
11. Determine the titer of excised phagemid (in cfu/ml) as follows:
⎡ Number of colonies ( cfu) × dilution factor ⎤

⎢ ⎥ × 1000 μl / ml
⎣ Volume of phagemid plated ( μ l ) ⎦
Mass Excision Results

Determine the excision efficiency as the ratio of the number of colony-
forming units rescued to the number of input lambda phage. Because the
excision efficiency is dependent on the ratio between the helper phage, the
phage stock, and the cells, the excision efficiency may vary.5, 17 If the
number of excised phagemid recovered is lower than expected when
performing mass excisions, repeat the excision with a higher number of
lambda phage and with freshly prepared XL1-Blue MRF´ cells.
At this point, single rescued colonies may be selected for plasmid preps and
DNA analysis.

Amplification of the Excised Phagemid Library
To generate the excised phagemid library, the supernatant containing the
excised phagemid particles from step 7 of the Mass Excision Protocol is
incubated with XLOLR host cells in the presence of ampicillin to produce a
stable amplified phagemid library.
Day 1
1. Grow an overnight culture (50 ml) of XLOLR cells, in LB broth with

supplements at 30°C.
Day 2 (Early)
2. Re-grow the cells to mid-log phase by adding 0.25 ml of the XLOLR

cells to 50 ml of LB broth with supplements, in a 250-ml flask.
Incubate the cells at 37°C, with shaking, until the culture reaches an
OD600 of 0.3–0.4.
3. Gently spin down the XLOLR cells (1000 × g). Resuspend the cells in
10 mM MgSO4 to an OD600 of 1.0 (8 × 108 cells/ml).
4. In a 2-liter flask, combine XLOLR cells with a portion of the excision

supernatant (from step 7 of the Mass Excision Protocol) at a minimum
cells-to-phagemid ratio of 10:1. (Assume an OD600 of 1.0 equals a cell
concentration of 8 × 108 cells/ml.) Amplify a portion of the excision
supernatant which represents at least 10-fold more clones than found in
the primary lambda library. Incubate the phagemids and cells at 37°C
for 15 minutes.
5. Add 500 ml of LB broth containing 100 μg/ml of ampicillin§. Incubate

with shaking at 37°C until an OD600 of 0.3–0.4 is reached. Do not
incubate the cells overnight.
6. Spin at 500 × g for 10 minutes to pellet the cells. Isolate the plasmid
DNA from the pelleted cells using any suitable method such as
alkaline lysis.
§

Single-Clone Excision Protocol
Day 1
1. Core the plaque of interest from the agar plate and transfer the plaque
to a sterile microcentrifuge tube containing 500 μl of SM buffer and
20 μl of chloroform. Vortex the microcentrifuge tube to release the
phage particles into the SM buffer. Incubate the microcentrifuge tube
for 1–2 hours at room temperature or overnight at 4°C. This phage
stock is stable for up to 6 months at 4°C.
2. Grow separate 50-ml overnight cultures of XL1-Blue MRF´ and

XLOLR cells in LB broth with supplements at 30°C.
Day 2
3. Gently spin down the XL1-Blue MRF´ and XLOLR cells (1000 × g).
Resuspend each of the cell pellets in 25 ml of 10 mM MgSO4. Measure
the OD600, then adjust the cell concentration to an OD600 of 1.0
(8 × 108 cells/ml) in 10 mM MgSO4.
4. Combine the following components in a 14-ml BD Falcon™

polypropylene round-bottom tube:
200 μl of XL1-Blue MRF´ cells at an OD600 of 1.0

250 μl of phage stock (containing >1 × 105 phage particles)
1 μl of the ExAssist helper phage (>1 × 106 pfu/μl)
Note Briefly spin the lambda phage stock to ensure that the
chloroform is separated completely before removing the
aliquot used in the excision reaction.
5. Incubate the 14-ml BD Falcon polypropylene tube at 37°C for 15

minutes to allow the phage to attach to the cells.
6. Add 3 ml of LB broth with supplements and incubate the BD Falcon

polypropylene tube for 2.5–3 hours at 37°C with shaking. Because
clonal representation is not relevant, single-clone excision reactions can
be safely performed overnight.
Note The turbidity of the media is not indicative of the success of

the excision.
7. Heat the BD Falcon polypropylene tube at 65–70°C for 20 minutes to

lyse the lambda phage particles and the cells. Spin the tube at 1000 × g
for 15 minutes to pellet the cell debris.

8. Transfer the supernatant into a sterile BD Falcon polypropylene tube.
This stock contains the excised pAD-GAL4-2.1 phagemid packaged as
filamentous phage particles. (This stock may be stored at 4°C for
1–2 months.)
9. To plate the excised phagemids, add 200 μl of freshly grown XLOLR

cells from step 3 (OD600 = 1.0) to two 1.5-ml microcentrifuge tubes.
Add 100 μl of the phage supernatant (from step 8) to one
microcentrifuge tube and 10 μl of the phage supernatant to the other
microcentrifuge tube.
10. Incubate the microcentrifuge tubes at 37°C for 15 minutes.
11. Plate 200 μl of the cell mixture from each microcentrifuge tube on
LB–ampicillin (100 μg/ml) agar plates and incubate the plates
overnight at 37°C.
Due to the high-efficiency of the excision process, it may be necessary to

titrate the supernatant to achieve single-colony isolation.
Colonies appearing on the plate contain the pAD-GAL4-2.1 double-stranded

phagemid with the cloned DNA insert. Helper phage will not grow, since
helper phage is unable to replicate in the Su– (nonsuppressing) XLOLR
strain and does not contain ampicillin-resistance genes.
To maintain the pAD-GAL4-2.1 phagemid, streak the colony on a new

LB–ampicillin agar plate. For long-term storage, prepare a bacterial glycerol
stock and store at –80°C.

DNA-BINDING DOMAIN VECTOR CONSTRUCTION
Bait Protein Insert Preparation, Ligation, and Transformation
DNA encoding the bait protein is prepared for insertion into the pBD-GAL4
Cam phagemid vector either by restriction digestion or PCR amplification.
DNA encoding the bait protein must be inserted so that the bait protein is
expressed in the same reading frame as the GAL4 BD (Figure 5). In the
MCS of the pBD-GAL4 Cam phagemid vector, the EcoR I, Srf I, Sal I, and
Pst I sites are unique; however, the Xho I, Sma I, and Xba I sites are not.
In addition, the Xba I site contains the UAG amber suppressor in the same
translational reading frame as the GAL4 domain. DNA should therefore be
inserted such that the Xba I site is not between the GAL4 domain and the
DNA insert.
Stratagene suggests dephosphorylation of the digested pBD-GAL4 Cam

phagemid vector with CIAP prior to ligating to the insert DNA. If more than
one restriction enzyme is used, the background can be reduced further by
selective precipitation using ammonium acetate, eliminating the small
fragment that appears between the two restriction enzyme sites.
1. Digest 5 μg of the pBD-GAL4 Cam phagemid vector in a final volume

of 50 μl.
2. Extract with an equal volume of phenol–chloroform until a clear

interface is obtained.
3. Repeat the extraction once with an equal volume of chloroform only.
4. Add an equal volume of 4 M NH4OAc to the aqueous phase.
5. Add 2.5 volumes of 100% (v/v) ethanol equilibrated at room

temperature. Immediately spin in a microcentrifuge at room
temperature to precipitate the vector DNA.
6. Wash the pellet once with 70% (v/v) ethanol.

7. Resuspend the pellet in a volume of TE buffer§ that will allow the
concentration of the vector DNA to be the same as the concentration of
the insert DNA (~0.1 μg/μl).
For ligation, the ideal ratio of insert-to-vector DNA is variable; however, a

reasonable starting point is 1:1 (insert-to-vector molar ratio), measured in
available picomole ends. This is calculated as follows:
2 × 10 6
Picomole ends / microgram of DNA =
number of base pairs × 660
§

Stratagene suggests the following protocol which includes three control
ligations:
Ligation Reaction Experimental Control

Components 1a 2a 3b 4c 5d
Prepared vector (0.1 μg/μl) 1.0 μl 1.0 μl 1.0 μl 1.0 μl 0 μl
Prepared insert (0.1 μg/μl) X μl X μl × 2 0 μl 0 μl 1.0 μl
10 mM rATP (pH 7.0) 1.0 μl 1.0 μl 1.0 μl 1.0 μl 1.0 μl
10× ligase buffer§ 1.0 μl 1.0 μl 1.0 μl 1.0 μl 1.0 μl
T4 DNA ligase (4 U/μl) 0.5 μl 0.5 μl 0.5 μl 0 μl 0.5 μl
Double-distilled water (to 10 μl) Y μl Y μl X μl X μl X μl
a
Experimental samples 1 and 2 vary the insert-to-vector ratio.
b
Control sample 3 tests to ensure the effectiveness of the digestion and CIAP treatment of
the vector.
c
Control sample 4 tests to ensure the vector was cleaved completely or if residual uncut
vector remains.
d
Control sample 5 tests to ensure the insert alone is not contaminated with the
vector DNA.
1. Ligate overnight at 4°C. When using blunt ends, ligate overnight at

12–14°C.
2. Transform 1–5 μl of the ligation mix into the appropriate competent

bacteria. Plate on selective media. See Table VI for expected results.
TABLE VI
Expected Results
Amount of Expected
transformation colony Efficiency
Sample plated number (cfu/μg of DNA)
Sample 1 (experimental) ≤200 μl will vary a will vary
Sample 2 (experimental) ≤200 μl will vary a
will vary
Sample 3 (control) ≤200 μl low numberb —
Sample 4 (control) ≤200 μl no colonies c
—
Sample 5 (control) ≤200 μl no colonies d
—
a
These plates represent recombinants.
b
This plate should have low numbers of colonies if the digestion and CIAP treatment
were effective.
c
This plate should have no colonies if the digest was complete.
d
This plate should have no colonies if the insert did not contain vector DNA.
§

Select isolated colonies for miniprep analysis to identify transformed
colonies containing the pBD-GAL4 Cam phagemid vector with the DNA
insert. The nucleotide sequence of the DNA insert should be determined to
verify that the DNA insert will be expressed as a fusion protein with the
GAL4 BD and that the DNA insert does not contain mutations. The
following oligonucleotide primers can be used to determine the nucleotide
sequence of the DNA insert:
BD primer Oligonucleotide sequence

5´-BD primer 5´-GTGCGACATCATCATCGGAAG-3´
3´-BD primer 5´-CCTAAGAGTCACTTTAAAATT-3´
Expression of the bait protein may be verified by Western blot analysis with
an antibody that immunoreacts with either the protein expressed from the
DNA insert or the GAL4 BD. However, if the antibody used fails to detect
expression of the bait protein, it may not indicate that the bait protein is not
expressed. The ability of the antibody to detect the bait protein is dependent
on several factors including the affinity of the antibody for the bait protein
and the expression level of the bait protein.
Relatively low levels of expression of bait proteins may be advantageous.

Only the number of bait proteins required to bind to the UASGAL4 or
UASGAL1 in the yeast chromosome is needed. Overexpression of a toxic bait
protein can inhibit cell growth and even be lethal. Over-expression of the
bait protein can also result in a phenomenon known as “squelching.” When
squelching occurs, excess unbound bait proteins bind to the target proteins
thereby preventing the target proteins from interacting with the bait proteins,
which are bound to the UAS. Consequently, transcription of the reporter
genes is not activated and interacting proteins are not detected.
Yeast Transformation and Assay for Expression of Reporter Genes

The pBD-GAL4 Cam phagemid vector containing DNA encoding the bait
protein (bait plasmid) must be transformed into the yeast host and assayed
for expression of the lacZ and HIS3 reporter genes (described in Yeast
Transformation Protocols and Screening). If the bait plasmid is capable of
inducing expression of the lacZ and HIS3 reporter genes in the absence of
the pAD-GAL4-2.1 phagemid vector containing an insert, the bait plasmid
is unsuitable for detecting protein–protein interactions in the HybriZAP-2.1
two-hybrid vector system. Expression of the reporter genes by the bait
plasmid may occur if the bait protein is a transcriptional activator or
contains a region of amino acids which are highly acidic and are capable of
binding transcription factors in the yeast host. Deletion of portions of the
bait protein may eliminate expression of the reporter genes but may also
eliminate portions of the protein required for interaction.

YEAST TRANSFORMATION
Notes A number of specialized media and reagents are required for the
protocols in this section and in the Screening and Verification of
Interaction sections that follow. Please consult the Two-Hybrid
Vector System Media and Reagents subsection of Preparation of
Media and Reagents for detailed recipes and instructions for
preparation of the appropriate media and reagents.
TABLE VII
Selective Media for Yeast Transformations
Selective media
SD agar
Yeast transformations SD medium Transformation Interaction
Control plasmids
pGAL4 Without Leu Without Leu —
pBD-WT Without Trp Without Trp —
pAD-WT Without Leu Without Leu —
pBD-MUT Without Trp Without Trp —
pAD-MUT Without Leu Without Leu —
pLamin C Without Trp Without Trp —
pBD-WT and pAD-WT Without Leu Without Leu and Without Leu,
and Trp Trp Trp, and His
pBD-MUT and Without Leu Without Leu and Without Leu,
pAD-MUT and Trp Trp Trp, and His
pLamin C and Without Leu Without Leu and Without Leu,
pAD-WT and Trp Trp Trp, and His
pLamin C and Without Leu Without Leu and Without Leu,
pAD-MUT and Trp Trp Trp, and His
Bait plasmid Without Trp Without Trp —
Bait and target plasmids Without Leu Without Leu and Without Leu,
and Trp Trp Trp, and His
Simultaneous vs. Sequential Transformation of the Bait and Target

Plasmids
The bait and target plasmids can be introduced into the yeast strain either
simultaneously or sequentially. Stratagene recommends sequential
transformation. In sequential transformation, yeast are transformed first with
the bait plasmid as described in Small-Scale Transformation of Yeast
Competent Cells. Second, yeast competent cells containing the bait plasmid
are prepared and transformed with the target plasmid(s) as described in
Large-Scale/Experimental Transformation and assayed for expression of
reporter genes as described in Screening.

Simultaneous transformation of the bait and target plasmids is an alternative
to sequential transformation. Simultaneous transformation is especially
useful when the bait plasmid is toxic to the yeast cells thereby increasing the
difficulty of preparing competent cells containing the bait plasmid and
generates results 5 days faster than sequential transformation.9 Toxicity of
the bait protein can be determined by comparing growth curves of the
YRG-2 yeast strain containing the bait plasmid and YRG-2 yeast strain
containing the pBD-WT or pBD-MUT bait plasmid when grown in selective
media.
Control Plasmids
Stratagene recommends transforming the control plasmids into the YRG-2
strain prior to the initial transformation of the bait and target plasmids and
concurrently with all subsequent transformations of the bait and target
plasmids. The control plasmids are used separately or in pairwise
combination to transform YRG-2 yeast as outlined in Table VII and in
Preparation of Yeast Competent Cells and Small-Scale Transformation of
Yeast Competent Cells.
Yeast Transformation Protocol

Notes Sterile technique must be used throughout the Yeast
Transformation Protocol.
Stratagene recommends the use of wide-bore pipet tips when

pipetting yeast competent cells to reduce the shear forces
associated with standard pipet tips.
Competent cells should be used immediately after preparation.
Preparation of Yeast Competent Cells
1. Prepare a yeast culture as follows:
a. Inoculate 1 ml of YPAD broth in a 1.5-ml microcentrifuge tube

with two to four YRG-2 yeast colonies that are 2–3 mm in
diameter and no more than 1 week old. Vortex the culture
vigorously until no cell clumps are visible.
b. In a 250-ml flask, add the 1 ml of the yeast culture to 50 ml of

YPAD broth.
c. Incubate the diluted culture for 18–24 hours at 30°C with shaking
at 225–250 rpm.
d. Check the OD600. If the OD600 is ≥1.2, continue with step 2. If the
OD600 is <1.2, return the flask to the incubator for 1–2 hours and
then check the OD600 again. If OD600 is <1.2 after 24 hours, restart
culture with new colonies.
2. Add the 50-ml yeast culture to 300 ml of YPAD broth in a

1- or 2-liter flask.

3. Incubate the culture for 3 hours at 30°C with shaking at 225–250 rpm.
4. Harvest the cells by centrifugation at 1000 × g for 5 minutes at room

temperature.
5. Discard the supernatant and resuspend the cells in 50 ml of deionized

water.
6. Centrifuge the cells at 1000 × g for 5 minutes at room temperature.
7. Discard the supernatant and resuspend the cells in 1.5 ml of freshly

prepared TE–LiAc solution (see Preparation of Media and Reagents).
Small-Scale Transformation of Yeast Competent Cells
Note Each transformation requires one sterile 1.5-ml microcentrifuge

tube.
1. Prepare the carrier DNA (salmon sperm DNA at 20 mg/ml) by boiling

the salmon sperm DNA for 20 minutes. Chill the salmon sperm DNA
on ice.
2. Using wide-bore pipet tips, aliquot 100 μl of competent yeast cells per
3. Add 100 μg of carrier DNA to each tube.
4. Add 100 ng of the desired plasmid to each tube; for pairwise

transformations, add 200 ng of each plasmid for a total of 400 ng of
plasmid DNA in each tube.
5. Add 600 μl of TE–LiAc–PEG solution (see Preparation of Media and

Reagents) to each tube and mix the contents by vortexing.
6. Incubate the samples at 30°C for 30 minutes with shaking at 200 rpm.
7. Add 70 μl of DMSO to each tube and mix the contents gently.
8. Heat-shock the samples for 15 minutes in a 42°C water bath.
9. Place the tubes on ice for 10 minutes.
10. Centrifuge the samples at 3000 rpm for 10 seconds to pellet the cells.
11. Using standard pipet tips, carefully remove all of the supernatant from
the tubes. If necessary after removing the supernatant, spin the tubes in
a microcentrifuge for a few seconds, and using a pipet, remove any
residual supernatant.

12. Add 0.5 ml of 1× TE buffer to each tube and vortex the tube to
resuspend cells. If pipetting is required to resuspend cells, use of a
wide-bore pipet tip is recommended to reduce the shearing stress on the
yeast cells.
13. Using wide-bore pipet tips, plate the transformed cells on the
appropriate SD-selective plates. For single transformations, plate
150 μl of the transformed cells on each 100-mm plate. For
cotransformations, plate 125 μl of the transformed cells on each of two
100-mm plates.
14. Incubate the plates at 30°C for 2–4 days until colonies appear.
Proceed with the filter lift assay described in Screening to confirm the
interactions outlined by the expected results in Tables VIII and IX.
TABLE VIII
Expected Results for the Yeast Transformation Controls
Expected resultsa
SD agar SD agar SD agar plates
Yeast transformation plates w/o plates w/o w/o Leu and
Leu Trp Trp
pGAL4b Growth, blue
pBD-WT Growth, white
pAD-WT Growth, white
pBD-MUT Growth, white
pAD-MUT Growth, white
pLamin C Growth, white
pBD-WT and pAD-WT Growth, blue
pBD-MUT and pAD-MUT Growth, light blue
pLamin C and pAD-WT Growth, white
pLamin C and pAD-MUT Growth, white
Bait plasmid Growth, white
a
When plated on the selective medium and assayed for expression of the lacZ reporter
gene.
b
The expected transformation efficiency of the pGAL4 control plasmid may be as much
as 10-fold lower than the expected transformation efficiencies of the other control
plasmids.

TABLE IX
Expected Results for Interactions Between Control Plasmids
Yeast Purpose of
transformation control SD medium Expected Resulta
pBD-WT and Positive SD agar plates w/o Growth, blue
pAD-WT interaction Leu, Trp, and His
pBD-MUT and Positive SD agar plates w/o Growth, light blue
pAD-MUT interaction Leu, Trp, and His
pLamin C and Negative SD agar plates w/o No growth
pAD-WT interaction Leu, Trp, and His
pLamin C and Negative SD agar plates w/o No growth
pAD-MUT interaction Leu, Trp, and His
pGAL4 Positive control SD agar plates w/o Leu Growth, blue
for lacZ
expression
pBD-WT Negative SD agar plates w/o Trp Growth, white
control for lacZ
expression
a
When assayed for expression of the lacZ reporter gene.
Large-Scale/Experimental Transformation
Note Control transformations should be performed concurrently with

experimental transformations. For the control transformations,
use the protocols in Preparation of Yeast Competent Cells and
Small-Scale Transformation of Yeast Competent Cells.
Prepare yeast competent cells containing the bait plasmid for transformation
with target plasmid(s) according to the protocol in Preparation of Yeast
Competent Cells. This protocol prepares enough competent cells for one
transformation and can be adjusted for the number of transformations to be
performed. Incorporate the following modifications into the protocol:
♦ In step 1a, inoculate 1 ml of SD medium lacking Trp with yeast colonies

containing the bait plasmid.
♦ In step 1b, add 1 ml of the culture of yeast cells containing the bait
plasmid to 50 ml of SD medium lacking Trp.
♦ In step 2, add the 50 ml of the yeast cells containing the bait plasmid to
300 ml of SD medium lacking Trp.
♦ In step 3, grow the yeast cells in selective medium at 30°C with shaking
at 225–250 rpm until the OD600 reaches approximately 0.5.

Transfect the target plasmid(s) into the prepared yeast competent cells
containing the bait plasmid according to the protocol in Small-Scale
Transformation of Yeast Competent Cells, incorporating the following
modifications to scale up the transformation:
♦ In step 2, add 1 ml of yeast competent cells containing the bait plasmid

to each 50-ml conical tube.
♦ In step 3, add 2 mg of carrier DNA to each tube.
♦ In step 4, add 40 μg of target plasmid(s) to be transfected to each tube.
♦ In step 5, add 6 ml of TE–LiAc–PEG solution to each tube and vortex

the tubes to mix the contents.
♦ In step 7, add 700 μl of DMSO to each tube.
♦ In step 10, centrifuge the samples at 1000 × g for 5 minutes.
♦ In step 12, add 10 ml of 1× TE buffer to each tube.
♦ In step 13, spread 1, 10, and 100 μl of the transformed cells on SD agar
plates lacking Leu and Trp. Spread 1 μl of the transformed cells on an
SD agar plate lacking Leu and 1 μl on an SD and agar plate lacking Trp.
Spread the remaining transformed cells on SD agar plates lacking His,
Leu, and Trp at 250 μl of transformation/100-mm plate.
Confirmation of Protein–Protein Interaction

Colonies that grow on SD agar plates without His, Trp, and Leu are either
due to the leaky expression of the HIS3 reporter gene or to the specific
interaction between the bait and target proteins resulting in expression of the
HIS3 gene. To distinguish between leaky expression and specifically
interacting proteins, detection of the expression of the second reporter gene
(lacZ) is determined by the filter lift assay described in Screening.
SCREENING
Note A number of specialized media and reagents are required for the
protocols in the Screening and Verification of Interaction sections.
Please consult the Two-Hybrid Vector System Media and
Reagents subsection of Preparation of Media and Reagents for
detailed recipes and instructions for preparation of the
appropriate media and reagents.

Filter Lift Assay
Notes Do not try to bypass the filter lift assay by simply adding 5-bromo-
4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) directly to the
plate. Addition of X-gal directly to the plate will inhibit yeast
cell growth.
Wear gloves and use sterile technique throughout the Filter Lift
Assay. Handle the qualitative filter papers carefully as the papers
tend to tear easily when wet.
Colonies are transferred to filter paper, permeabilized in liquid nitrogen, and

assayed for expression of the lacZ reporter gene by the detection of
β-galactosidase activity with a solution containing an X-gal substrate.
Colonies producing β-galactosidase turn blue in color.
Note Nitrocellulose paper can be substituted for filter paper. White

colonies will eventually turn blue on filter paper, but colonies will
maintain their blue or white color on nitrocellulose paper.
1. Allow the transformants from step 14 of the Transforming Yeast

Competent Cells to grow for 3–7 days or until the colonies are 1–2 mm
in diameter.
2. Prepare the Z buffer with X-gal (see Preparation of Media

and Reagents).
3. Add 2 ml of Z buffer with X-gal to the bottom of a 100-mm petri dish.

Add a sterile qualitative filter paper to the dish (see Equipment in
Additional Materials Required). Ensure that the filter paper is
completely wet. Excess buffer should be poured off into a
waste beaker.
Note If the transformations were plated on 150-mm plates, use

4.5 ml of Z buffer and 150-mm petri dishes.
4. Label a separate piece of sterile filter paper. Hold the paper with
forceps and starting from the edge of the paper, slowly place the filter
on the plate. Ensure that the filter paper contacts all of the colonies on
the plate; allow contact for approximately 1 minute. Mark the
orientation on the plate and on the filter.
5. Using forceps and starting at one side of the plate, carefully lift the
filter paper from the plate.
6. Holding the filter paper with forceps, dip the paper colony side up in
liquid nitrogen for ten seconds. Remove the filter paper from the liquid
nitrogen and allow it to thaw (colony side up). Repeat this step two or
three times with each filter paper.

7. Carefully place the thawed filter paper colony side up onto the filter
paper soaked in the Z buffer with X-gal (see step 3). Carefully remove
any air bubbles trapped between the two pieces of filter paper.
8. Allow the plates containing the filter papers to incubate at room

temperature for 3 hours. During the incubation, the colonies containing
the pGAL4 control will turn blue. The pAD-WT and pBD-WT
cotransformants will turn a similar shade of blue. The pAD-MUT and
pBD-MUT cotransformants will turn light blue. No color change
should be observed in the pAD-WT and the pLaminC cotransformants
(see Table VIII).
Note Colonies containing the pGAL4 control plasmid will be a

more intense blue color than colonies containing the positive
control plasmids. The pGAL4 control plasmid expresses the
complete GAL4 protein and activates transcription of the
lacZ reporter gene more efficiently than the portions of the
GAL4 protein that are reconstituted by the interacting cI-wt
or cI-E233K protein. The most important factor in evaluating
the color of the yeast colonies containing control plasmids is
whether the blue color of the cI-wt or cI-E233K-containing
colony can be distinguished from the color of the pAD-WT or
pAD-MUT and pLamin C-containing colonies.
9. Colonies with β-galactosidase activity can be isolated by aligning the

filter paper and the plate. Colonies should be streaked again on a new
plate with selective media to select for His+ colonies. Repeating this
assay to verify the presence of β-galactosidase activity in LacZ+
colonies is recommended.
VERIFICATION OF INTERACTION
Isolation of Plasmid DNA from Yeast
Plasmid DNA can be isolated from yeast in sufficient quality and quantity to
transform E. coli either by using the Yeast DNA Isolation System or by
following this quick and easy procedure.18 This procedure yields a mixture
of intact plasmid DNA and fragmented chromosomal DNA; therefore, the
resultant plasmid DNA is not of sufficient purity for gel analysis.
1. Inoculate 2 ml of YPAD broth§ with an isolated His+–LacZ+ yeast

colony. Incubate the culture at 30°C until the media is saturated
(~2–3 days).
2. Transfer the yeast culture to a 1.5-ml microcentrifuge tube and spin at

14,000 × g for 10 seconds to pellet the yeast cells. Decant the
supernatant.
§

3. Add 0.2 ml of yeast lysis solution§ and resuspend the yeast cells by
vortexing. Add 0.2 ml of phenol–chloroform–isoamyl alcohol§
[25:24:1 (v/v/v)] and 0.3 g of acid-washed glass beads. Vortex the
suspension for 2 minutes.
4. Spin the suspension at 14,000 × g for 5 minutes at room temperature.

Transfer the top aqueous phase containing the DNA to a new
5. Precipitate the DNA with 1/10 volume of 3 M NaOAc (pH 5.2) and
2.5 volumes of ethanol. Spin the suspension at 14,000 × g for
10 minutes. Decant the supernatant.
6. Wash the DNA pellet with 1 ml of 70% (v/v) ethanol and re-spin the
pellet at 14,000 × g for 10 minutes. Decant the supernatant and dry the
DNA pellet under a vacuum.
7. Resuspend the DNA pellet in 50 μl of TE buffer. Use 5–20 μl to

transform the XL1-Blue MRF´ competent cells and select for the target
or bait plasmid by plating on LB–ampicillin or LB–chloramphenicol§
agar plates, respectively.
8. Identify colonies that contain the target or bait plasmid by preparing

miniprep DNA from isolated colonies from the LB–ampicillin or
LB–chloramphenicol agar plates, respectively, and by restriction digest
analysis.
Analysis by PCR
Target DNA inserts can be analyzed by PCR using the following AD
primers:
AD Primer Oligonucleotide sequence

5´-AD primer 5´-AGGGATGTTTAATACCACTAC-3´
3´-AD primer 5´-GCACAGTTGAAGTGAACTTGC-3´
The pAD-WT control plasmid is the positive control for this reaction. PCR
with the pAD-WT control plasmid and the AD primers yields a 0.5-kb PCR
product.
§

TABLE X
Verification of the Transformation of the Bait and Target
Plasmids
AD vector BD vector Selective medium Expected resulta
Target vector — SD agar plate without Growth, white
Leu
Target vector pBD-WT SD agar plate without Growth, white
Leu and Trp
Target vector pBD-MUT SD agar plate without Growth, white
Leu and Trp
Target vector pLamin C SD agar plate without Growth, white
Leu and Trp
Target vector pBD-GAL4 Cam SD agar plate without Growth, white
Leu and Trp
Target vector Bait vector SD agar plate without Growth, blue
Leu and Trp
a
When transformed into YRG-2, plated on the selective medium and assayed for
expression of the lacZ reporter gene.
TABLE XI
Verification of the Specificity of the Interaction between the Bait
and Target Proteins
AD vector BD vector Selective medium Expected resulta
Target vector pBD-WT SD agar plate without No growth
Leu, Trp, and His
Target vector pBD-MUT SD agar plate without No growth
Leu, Trp, and His
Target vector pLamin C SD agar plate without No growth
Leu, Trp, and His
Target vector pBD-GAL4 Cam SD agar plate without No growth
Leu, Trp, and His
Target vector Bait vector SD agar plate without Growth, blue
Leu, Trp, and His
a
When transformed into YRG-2, plated on the selective medium and assayed for
expression of the lacZ reporter gene.

Verification of Specificity of Protein–Protein Interactions
To verify the specificity of the interaction between the bait and target
proteins, transform yeast and plate on selective media as indicated in Tables
X and XI. Assay the transformants for expression of the HIS3 and lacZ
reporter genes and compare the results of the assay to the
expected results.
1. Transform the yeast competent cells as described in the Yeast

Transformation Protocols subsection of Yeast Transformation.
2. Streak the transformants that grow on the SD agar plates without Leu
and Trp onto SD agar plates without Leu, Trp, and His. Incubate the
plates for 3–7 days at 30°C.
3. Determine expression of the lacZ gene of the cotransformants from

step 2 by the filter lift assay described in Screening.
If the transformants do not give the expected results, see Troubleshooting.

For additional discussion regarding false positives, see References 15, 19
and 20.15, 19, 20
To identify the protein encoded by the target DNA, the nucleotide sequence
of the target DNA can be determined and compared to protein and
nucleotide sequence databases to identify related or homologous proteins.
The target DNA can also be used as a hybridization probe to screen the
lambda library for full-length target DNA clones and for clones with high
homology to the target DNA.
The DNA insert encoding the target protein can be transferred from the
pAD-GAL4-2.1 vector to a protein expression/purification vector by
digesting the vector with BamH I, Nhe I, or EcoR I restriction enzymes at
the 5´ end of the DNA insert and with Xho I, Sal I, Xba I, or Bgl II
restriction enzymes at the 3´ end of the DNA insert. Prokaryotic expression
vectors having compatible restriction sites include the pCAL-n and
pCAL-n-EK vectors and the pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, and
pGEX-5X-1 vectors (available from Amersham Biosciences, Piscataway,
New Jersey). A eukaryotic expression vector having compatible restriction
sites is the pESP-2 vector. The pCAL-n and pCAL-n-EK vectors express the
target protein as a fusion protein with the calmodulin peptide and the
pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-5X-1, and pESP-2 vectors
express the target protein as a fusion protein with glutathione-s-transferase
(GST). These fusion proteins can then be used in in vitro
immunoprecipitation assays with the bait protein.
Discussion regarding further verification of protein–protein interactions can

be found in numerous publications.15, 21

APPENDIX: GENERAL COMPARISON OF Escherichia coli VERSUS
YEAST HOST STRAINS
Host strain
Quality/feature Escherichia coli Yeast
Doubling time 20 minutes >1 hour
Complex media (nonselective) LB and NZY YPAD
Chemically defined media (selective) M9 SD
pH 7 (neutral) 5.8 (acidic)
Growth temperature 37°C 30°C
Antibiotic sensitivity Sensitive to most antibiotics Resistant to most antibiotics including
ampicillin
Selection method for presence of plasmid Add antibiotic to media Remove amino acid from media
Colonial morphology Small, flat colonies Large, rounded colonies
Cell diameter 1 μm 3–5 μm
Odor Musty, pungent Bread dough

TROUBLESHOOTING
Mass Excision
Observation Suggestion
r
The number of Amp colonies in the All of the host cells are not infected with helper phage during the mass excision,
mass excision are greater allowing lambda phage to replicate lytically before conversion to the phagemid.
than expected Verify the titer of lambda phage and helper phage prior to the mass excision;
infect the host cells with helper phage prior to growing to mid log phase on Day 2
(cells infected with the helper phage will grow more slowly than uninfected cells).
Absence of Ampr colonies Ensure that XL1-Blue MRF´ host cells are used for the mass excision.
Two-Hybrid Vector System Screening

The bait protein is not detected in Verify the nucleotide sequence of the GAL4 BD and the insert DNA to ensure that
Western blot analysis they are in the same frame.
Ensure the insert DNA is expressed at levels sufficient to be detectable with the
antibody used. To activate transcription of the reporter genes, only a sufficient
number of bait proteins to bind to all of the GAL4 operators is required; therefore,
a low level of bait protein may be adequate in the two-hybrid assay. If the
nucleotide sequence encoding the bait protein is correct, continue with the two-
hybrid screening.
If the antibody does not have a sufficiently high affinity for the bait protein, the bait
protein may be expressed but may not be detectable. If the nucleotide sequence
encoding the bait protein is correct, continue with the two-hybrid screening.
Transformation with the bait Subclone portions of the bait protein (see Yeast Transformation and Assay for
plasmid alone results in Expression of Reporter Genes) to ensure that bait protein alone does not activate
His+–LacZ+ colonies transcription of the reporter genes.
The control plasmids do not give Verify that correct control plasmid pairs are used.
the expected results Prepare a fresh solution of Z buffer with X-gal.
Incubate the qualitative filter paper colony side up during color development to
avoid smearing of the colonies.
Ensure that Z buffer and X-gal solutions are prepared correctly.
Avoid exceeding the recommended amount of Z buffer with X-gal; excessive
amounts may cause smearing of colonies.
Verify the pH of the SD agar plates using a pH indicator strip.
Verify the phenotype of a yeast colony as described in Yeast Host Strain Phenotype
and prepare new yeast competent cells using the same yeast colony.
Use sterile technique when preparing and transforming the yeast competent cells
to avoid contamination with a different yeast strain or E. coli.
Ensure that the filter paper makes good contact with the yeast colonies.
Verify that the colony material has been transferred by visual inspection of the filter
paper and the colonies on the plate.
The transformants of the pBD-WT If the colonies do not contain both control plasmids, blue color will not be
and pAD-WT pair of control observed. Verify that medium was made correctly to select for both control
plasmids or the pBD-MUT and plasmids.
pAD-MUT pair of control plasmids Verify that the correct control plasmid pair is used.
do not turn blue
(table continues on the next page)

(table continues from the previous page)
The transformants of the pLamin C Verify that the correct control plasmid pair is used.
and pAD-WT pair of control
plasmids or the pLamin C and pAD-
MUT pair of control plasmids turn
blue
No His+–LacZ+ transformants Verify the nucleotide sequence of the GAL4 BD and insert DNA to ensure the bait
are present protein is expressed.
The frequency of target proteins in the library may be low. Prepare and screen
additional cotransformants; screen a different library.
To verify that the screening procedure is working, screen a library in which
expression of the bait protein is known.
Verify the pH of the SD agar plates using a pH indicator strip.
Vary the fusion point of the GAL4 BD and the bait protein to highlight possible
steric inhibition.
Verify that the competent cells containing the bait plasmid were used to transform
the target plasmid(s).
For good transfer of yeast colonies, ensure that the filter paper makes good
contact with the yeast colonies.
Verify that the colony material has been transferred by visual inspection of the filter
paper and the colonies on the plate.
The colonies are small and are Visually compare the E. coli and yeast on the plate, noting that E. coli colonies are
His+–LacZ+, but the colonies are small and flat and yeast are large, round, and white. If further comparison is
also flat and do not required, prepare a slide and view the E. coli and yeast under a microscope,
grow larger with noting that yeast are large and round and E. coli cells are small and rod shaped.
continued incubation
Plasmid Isolation from Yeast

Absence of Ampr or Camr colonies Transform E. coli with a greater volume of isolated DNA or reisolate plasmid DNA
when E. coli is transformed with to ensure that the transformation is performed with a sufficient amount of
DNA isolated from yeast plasmid DNA.
Continue incubation of the transformants to compensate for the slow growth rate
of the Camr transformants.
No discernible bands following Transform E. coli with plasmid DNA isolated from yeast before restriction analysis,
restriction analysis of the recovered as the plasmid DNA isolated may be contaminated with yeast chromosomal DNA
plasmid DNA and is not suitable for restriction analysis.

Verification of Interaction
+ +
Presence of His –LacZ colonies If the target protein is capable of initiating transcription of both reporter genes in
when the target plasmid alone is the absence of the bait protein, the target plasmid is a false positive and should
transformed into yeast be discarded.
Presence of His+–LacZ+ colonies If the target protein is capable of initiating transcription of both reporter genes in
when the target plasmid and the the presence of the BD vector irrespective of the expression of a bait protein, the
pBD-WT and pLamin C pair of target plasmid is a false positive and should be discarded.
control plasmids, the pBD-MUT and
pLamin C pair of control plasmids,
or the pBD-GAL4 Cam phagemid
vector is transformed into yeast
Absence of His+–LacZ+ colonies Determine if the original yeast host contained more than one target plasmid by
when target and bait plasmids are miniprep analysis of target plasmid DNAs.
transformed into yeast

PREPARATION OF MEDIA AND REAGENTS
Standard Media and Reagents
Note All media must be autoclaved prior to use.
LB Agar (per Liter) LB Broth (per Liter)

10 g of NaCl 10 g of NaCl
10 g of tryptone 10 g of tryptone
5 g of yeast extract 5 g of yeast extract
20 g of agar Adjust to pH 7.0 with 5 N NaOH
Adjust pH to 7.0 with 5 N NaOH Add deionized H2O to a final volume
Add deionized H2O to a final volume of 1 liter
of 1 liter Adjust to pH 7.0 with 5 N NaOH
Adjust pH to 7.0 with 5 N NaOH Autoclave
Autoclave
Pour into petri dishes (~25 ml/100-mm
plate)
LB–Ampicillin Agar (per Liter) LB Broth with Supplements
1 liter of LB agar, autoclaved Prepare 1 liter of LB broth
Cool to 55°C Autoclave
Add 10 ml of 10-mg/ml filter-sterilized Add the following filter-sterilized
ampicillin supplements prior to use
Pour into petri dishes 10 ml of 1 M MgSO4
(~25 ml/100-mm plate) 3 ml of a 2 M maltose solution or 10 ml
of 20% (w/v) maltose
LB–Chloramphenicol Agar (per LB–Ampicillin Broth (per Liter)
1 liter of LB broth, autoclaved
Liter)
Cool to 55°C
Prepare 1 liter of LB agar
Add 10 ml of 10-mg/ml filter-sterilized
Autoclave
ampicillin
Cool to 55°C
Add 30 mg of filter-sterilized
chloramphenicol
Pour into petri dishes (~25 ml/100-mm
plate)
LB–Tetracycline Broth (per Liter) LB–Tetracycline Agar (per Liter)

Prepare 1 liter of LB broth Prepare 1 liter of LB agar
Autoclave Autoclave
Cool to 55°C Cool to 55°C
Add 12.5 mg of filter-sterilized tetracycline Add 12.5 mg of filter-sterilized tetracycline
Store broth in a dark, cool place as Pour into petri dishes (~25 ml/100-mm plate)
tetracycline is light-sensitive Store plates in a dark, cool place or cover
plates with foil if left out at room
temperature for extended time periods as
tetracycline is light-sensitive

10× Ligase Buffer NZY Agar (per Liter)
500 mM Tris-HCl (pH 7.5) 5 g of NaCl
70 mM MgCl2 2 g of MgSO4 . 7H2O
10 mM DTT 5 g of yeast extract
Note rATP is added separately in the 10 g of NZ amine (casein hydrolysate)
ligation reaction 15 g of agar
Add deionized H2O to a final volume of
TE Buffer 1 liter
10 mM Tris-HCl (pH 7.5) Adjust the pH to 7.5 with NaOH
1 mM EDTA Autoclave
Pour into petri dishes (~25 ml/100-mm plate
or ~80 ml/150-mm plate)
NZY Broth (per Liter) NZY Top Agar (per Liter)
5 g of NaCl Prepare 1 liter of NZY broth
2 g of MgSO4 . 7H2O Add 0.7% (w/v) agarose
5 g of yeast extract Autoclave
10 g of NZ amine (casein hydrolysate)
Add deionized H2O to a final volume of
1 liter
Adjust the pH to 7.5 with NaOH
Autoclave
SM Buffer (per Liter) Super Broth (per Liter)ll

5.8 g of NaCl 35 g of tryptone
2.0 g of MgSO4 · 7H2O 20 g of yeast extract
50.0 ml of 1 M Tris-HCl (pH 7.5) 5 g of NaCl
5.0 ml of 2% (w/v) gelatin Add deionized H2O to a final volume
Add deionized H2O to a final volume of 1 liter of 1 liter
Autoclave Adjust to pH 7.5 with 5 M NaOH
Autoclave
ll
LB broth is the medium of choice for overnight growth. However, when growing XL1-Blue MRF´ for in vivo excision,
rescue, or minipreps, super broth may be used. Growing host cells overnight and plating cultures at 30°C also increases
plating efficiency.

Two-Hybrid Vector System Media and Reagents
Media for Growth and Maintenance of Yeast
YPAD Medium
YRG-2 cells are grown on YPAD medium. YPAD is a rich medium and
does not select for yeast containing a plasmid. Yeast are streaked for
isolation on YPAD agar plates and are incubated at 30°C for 1–2 days until
colonies appear. Liquid YPAD broth is used for growing yeast for
transformation. Adenine sulfate is added to the medium to reduce the
reversion rate of the ade2-101 mutation thereby reducing the amount of
reddish pigment in the yeast colonies.
YPAD Agar (per Liter) YPAD Broth (per Liter)

20 g of Difco peptone 20 g of Difco® peptone
10 g of yeast extract 10 g of yeast extract
15–20 g of agar Add deionized H2O to a final
Add deionized H2O to a final volume of 960 ml
volume of 960 ml Adjust the pH to 5.8
Adjust the pH to 5.8 Add 40 mg of adenine sulfate
Add 40 mg of adenine sulfate Autoclave
Autoclave Cool to 55°C
Cool to 55°C Add glucose to 2% (v/v) by
Add glucose to 2% (v/v) by adding 40 ml of a 50%
adding 40 ml of a 50% stock solution which has
stock solution which has been filter sterilized or
been filter sterilized or autoclaved separately
autoclaved separately

Synthetic Minimal Medium
Synthetic minimal (SD) medium is used for selection of yeast containing a
plasmid. SD medium contains a yeast nitrogen base, a carbon source
[2% (w/v) glucose], and a dropout solution. The dropout solution contains
specific amino acids and other nutrients required for growth of the yeast.
The omission of Leu from SD medium selects for the pAD-GAL4-2.1
vector or the pAD-WT or pAD-MUT control plasmid, which contain the
LEU2 gene. The omission of Trp from SD medium selects for the
pBD-GAL4 Cam vector or the pBD-WT, pBD-MUT, or pLamin C control
plasmid, which contain the TRP1 gene. The omission of both Leu and Trp
from SD medium selects for both vectors or a pair of control plasmids. The
omission of Leu, Trp, and His from SD medium selects for both phagemid
vectors and for hybrid proteins that interact.
SD Agar (per Liter) SD Medium (per Liter)

6.7 g of Difco yeast nitrogen 6.7 g of Difco yeast nitrogen
base without amino acids base without amino acids
(Difco Catalog #0919-15-3) (Difco Catalog #0919-15-3)
182.2 g of D-sorbitol 182.2 g of D-sorbitol
15–20 g of agar Add deionized H2O to a final
Add deionized H2O to a final volume of 860 ml
volume of 860 ml. Adjust pH to 5.8
Adjust pH to 5.8 Autoclave
Autoclave Add 100 ml of the appropriate
Cool to 55°C 10× dropout solution (see 10×
Add 100 ml of the appropriate Dropout Solution) and 40 ml of
10× dropout solution (see 10× a 50% stock solution of
Dropout Solution) and 40 ml of glucose which has been filter
a 50% stock solution of sterilized or autoclaved
glucose which has been filter separately
sterilized or autoclaved
separately
Pour into 100- and 150-mm
petri dishes

10× Dropout Solution
To prepare the appropriate 10× dropout solution for the desired SD medium,
simply omit the appropriate component as indicated in the footnotes to the
table that follows. All amino acids and nutrients can be autoclaved with the
exception of tryptophan, threonine and aspartic acid which should be
filter sterilized. After sterilization, the 10× dropout solutions can be stored in
100-ml aliquots at 4°C for up to 1 year.
Component Weight (mg/liter) Sigma Catalog #

L-Isoleucine 300 I 2752
L -Valine 1500 V 0500
L -Adenine hemisulfate salt 200 A 9126
L -Arginine HCl 200 A 5131
L -Histidine HCl monohydratea 200 H 8125
L -Leucine b
1000 L 8000
L -Lysine HCl 300 L 5626
L -Methionine 200 M 9625
L -Phenylalanine 500 P 2126
L -Threonine 2000 T 8625
L -Tryptophanc 200 T 0254
L -Tyrosine 300 T 3754
L -Uracil 200 U 0750
L -Glutamic acid 1000 G 1251
L -Aspartic acid 1000 A 9256
L -Serine 4000 S 4500
a
Omit L-histidine HCl monohydrate for selection of interacting proteins.
b
Omit L-leucine for selection of the pAD-GAL4-2.1 phagemid vector or the pAD-WT or
pAD-MUT control plasmid.
c
Add these amino acids only after autoclaving the 10× dropout solution.
d
Omit L-tryptophan for selection of the pBD-GAL4 Cam phagemid vector or the pBD-WT,
pBD-MUT, or pLamin C control plasmid.

Yeast Transformation Solutions
Stock Solutions
The following stock solutions are necessary in order to prepare the yeast
transformation solutions outlined below:
10× Lithium Acetate (LiAc) 50% (w/v) PEG 3350

1 M LiAc (Sigma Catalog #L 6883) 50 g of PEG (Sigma
Adjust pH to 7.5 with dilute Catalog #P 3640,
acetic acid average molecular
Autoclave weight: 3350)
Store at room temperature dH2O to 100 ml
Filter sterilize or autoclave
Store at room temperature
TE–LiAc–PEG Solution 10× TE buffer
(1× TE buffer, 1× LiAc, 100 mM Tris-HCl
(pH 7.5)
40% (w/v) PEG 3350) 10 mM EDTA (pH 8.0)
1 ml of 10× TE buffer
Autoclave
1 ml of 10× LiAc
Store at room temperature
8 ml of 50% (w/v) PEG 3350
TE–LiAc Solution 1× TE Buffer

(1× TE buffer and 1× LiAc) 1 ml of 10× TE buffer
1 ml of 10× TE buffer 9 ml of dH2O
1 ml of 10× LiAc
8 ml of sterile dH2O
Salmon Sperm DNA

Sonicate or randomly shear the salmon sperm DNA. For higher efficiency,
phenol–chloroform extract and resuspend in TE buffer at a concentration of
20 mg/ml. Store the aliquots at –20°C. Before use, boil the salmon sperm
DNA for 5 minutes.

Solutions for the Filter Lift Assay
Z Buffer Stock Solution (per Liter)
16.1 g of Na2HPO4 ⋅ 7H2O
5.5 g of NaH2PO4 ⋅ H2O
0.75 g of KCl
0.246 g of MgSO4 ⋅ 7H2O
Add dH2O to a volume of 1 liter
Adjust the pH to 7.0
Autoclave or filter sterilize
Store at 4°C
Z Buffer with X-gal (100 ml) X-gal Stock Solution
Dissolve X-gal in N,N-
Note Prepare fresh each time dimethyl-formamide
(DMF) at a
98 ml of Z buffer concentration of
0.27 ml of β-mercaptoethanol 20 mg/ml
1.67 ml of X-gal stock solution Store at –20°C
Solutions for Plasmid DNA Isolation from Yeast

Phenol–Chloroform– Yeast Lysis Solution
Isoamyl Alcohol (100 ml) 2% (v/v) Triton® X-100
50 ml of neutralized phenol 1% (w/v) SDS
(neutralized with Tris-HCl as 100 mM NaCl
described in Reference 19) 10 mM Tris-HCl (pH 8.0)
48 ml of chloroform 1 mM EDTA
2 ml of isoamyl alcohol Store at room temperature
Store at 4°C

REFERENCES
1. Fields, S. and Song, O. (1989) Nature 340(6230):245-6.
2. Mullinax, R. L. and Sorge, J. A. (1995) Strategies 8(1):3-5.
3. Ma, J. and Ptashne, M. (1987) Cell 51(1):113-9.
4. Alting-Mees, M. A. and Short, J. M. (1989) Nucleic Acids Res 17(22):9494.
5. Short, J. M., Fernandez, J. M., Sorge, J. A. and Huse, W. D. (1988) Nucleic Acids Res
16(15):7583-600.
6. Short, J. M. and Sorge, J. A. (1992) Methods Enzymol 216:495-508.
7. Jerpseth, B., Greener, A., Short, J. M., Viola, J. and Kretz, P. L. (1992) Strategies
5(3):81–83.
8. Feilotter, H. E., Hannon, G. J., Ruddell, C. J. and Beach, D. (1994) Nucleic Acids Res
22(8):1502-3.
9. Callahan, M., Jerpseth, B., Mullinax, R. L. and Greener, A. (1995) Strategies 8(2):45–
46.
10. Guthrie, C. and Fink, G. R. (Eds.). (1991). Guide to Yeast Genetics and Molecular
Biology, Vol. 194. Academic Press, Inc., Boston.
11. Estojak, J., Brent, R. and Golemis, E. A. (1995) Mol Cell Biol 15(10):5820-9.
12. Ptashne, M., Backman, K., Humayun, M. Z., Jeffrey, A., Maurer, R. et al. (1976)
Science 194(4261):156-61.
13. Cohen, S., Knoll, B. J., Little, J. W. and Mount, D. W. (1981) Nature 294(5837):182-4.
14. Gimble, F. S. and Sauer, R. T. (1989) J Mol Biol 206(1):29-39.
15. Bartel, P. L., Chien, C.-T., Sternglanz, R. and Fields, S. (1993). Using the Two-hybrid
System to Detect Protein-Protein Interactions. In Cellular Interactions in Development:
A Practical Approach,D. A. Hartley (Ed.), pp. 153–179. Oxford Univ. Press, Oxford,
England.
16. Dotto, G. P., Horiuchi, K. and Zinder, N. D. (1984) J Mol Biol 172(4):507-21.
17. Hogrefe, H. H., Mullinax, R. L., Lovejoy, A. E., Hay, B. N. and Sorge, J. A. (1993)
Gene 128(1):119-26.
18. Hoffman, C. S. and Winston, F. (1987) Gene 57(2-3):267-72.
19. Bartel, P., Chien, C. T., Sternglanz, R. and Fields, S. (1993) Biotechniques 14(6):920-4.
20. Durfee, T., Becherer, K., Chen, P. L., Yeh, S. H., Yang, Y. et al. (1993) Genes Dev
7(4):555-69.
21. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K. and Elledge, S. J. (1993) Cell
75(4):805-16.

ENDNOTES
ExAssist®, HybriZAP®, Lambda ZAP®, and ZAP-cDNA® are registered trademarks of
Stratagene in the United States.
Difco® is a registered trademark of Difco Laboratories.
GenBank® is a registered trademark of the U.S. Department of Health and Human Services.
Parafilm® is a registered trademark of American Can Company.
Triton® is a registered trademark of Rohm and Haas Co.
Whatman® is a registered trademark of Whatman Ltd.
BD Falcon is a trademark of BD Biosciences.
VWRbrand is a trademark of VWR Scientific.
MSDS INFORMATION
The Material Safety Data Sheet (MSDS) information for Stratagene products is provided on Stratagene’s
website at http://www.stratagene.com/MSDS/. Simply enter the catalog number to retrieve any associated
MSDS’s in a print-ready format. MSDS documents are not included with product shipments.

57
Premade Libraries
QUICK-REFERENCE PROTOCOL
♦ Ligate the bait DNA insert into the pBD-GAL4 Cam phagemid ♦ Mass excise to form the pAD-GAL4-2.1 library (target plasmid)
vector
♦ Transform into yeast and assay for reporter gene expression ♦ Transform yeast containing the pBD-GAL4 Cam phagemid
vector (bait plasmid) with the pAD-GAL4-2.1 library
♦ Assay cotransformants for reporter gene expression
♦ Restreak putative positives and reassay for reporter gene expression
♦ Isolate plasmids from yeast and transform into E. coli
♦ Isolate the pAD-GAL4-2.1 phagemid vector (target plasmid)
♦ Cotransform the target plasmid with the pBD-GAL4 Cam control plasmids into yeast and assay for
expression of reporter genes
♦ Discard the target plasmids that induce expression of reporter genes with the pBD-GAL4 Cam
control plasmids
♦ Perform secondary assays with those target plasmids that do not induce expression of reporter genes with
the pBD-GAL4 Cam control plasmids
58

Hybrizap - 2.1 Two-Hybrid Libraries: Instruction Manual

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HybriZAP®-2.1 Two-Hybrid cDNA Library Kit

Revision A Copyright © 2006 by Stratagene.

HybriZAP®-2.1 Two-Hybrid Libraries 1

Salmon sperm DNA

Equipment and Supplies

2 HybriZAP®-2.1 Two-Hybrid Libraries

The HybriZAP®-2.1 two-hybrid vector system* (Figure 1), a eukaryotic

The HybriZAP-2.1 two-hybrid vector system is particularly useful for the

* U.S. Patent Nos. 5,283,173; 5,468,614; 5,128,256; and 5,286,636.

HybriZAP®-2.1 Two-Hybrid Libraries 3

1. Ligate the insert DNA

4. Coexpress the target and bait proteins in yeast

4 HybriZAP®-2.1 Two-Hybrid Libraries

The HybriZAP-2.1 lambda vector and the pAD-GAL4-2.1 phagemid vector

HybriZAP®-2.1 Two-Hybrid Libraries 5

HybriZAP®-2.1 Vector Map

Right End 45.52 kb

Hind III 27.17

Hind III 36.59

Hind III 41.15

A-J att int xis c1857 (nin5)

6 HybriZAP®-2.1 Two-Hybrid Libraries

Library Construction in the HybriZAP®-2.1 Vector

The adapters are comprised of 9- and 13-mer oligonucleotides, which are

HybriZAP®-2.1 Two-Hybrid Libraries 7

pAD-GAL4-2.1 Multiple Cloning Site Region

Xho I Sal I Xba I

Feature Nucleotide position

8 HybriZAP®-2.1 Two-Hybrid Libraries

pBD-GAL4 Cam Multiple Cloning Site Region

Feature Nucleotide position

HybriZAP®-2.1 Two-Hybrid Libraries 9

Bacterial Strain Genotypes

XLOLR Strain Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 thi-1 recA1

XL1-Blue MRF´ Bacterial Strain Description

Note The mcrA, mcrCB, and mrr mutations prevent restriction of

10 HybriZAP®-2.1 Two-Hybrid Libraries

Establishing an Agar Plate Bacterial Stock

2. Streak the splinters onto an LB agar plate containing the appropriate

3. Incubate the plate overnight at 37°C.

5. Restreak the colonies onto a fresh plate every week.

Preparation of a –80°C Bacterial Glycerol Stock

2. Add 4.5 ml of a sterile glycerol–liquid medium solution (prepared by

3. Aliquot into sterile centrifuge tubes (1 ml/tube).

HybriZAP®-2.1 Two-Hybrid Libraries 11

Preparation of the Plating Culture

1. Streak the XL1-Blue MRF´ bacterial glycerol stock onto

2. Inoculate 50 ml of LB broth with supplements in a sterile flask with a

Note For later use, store the cells at 4°C overnight in

6. Dilute the cells to an OD600 of 0.5 with sterile 10 mM MgSO4.

Note The bacteria should be used immediately following dilution.

Measuring the Titer

12 HybriZAP®-2.1 Two-Hybrid Libraries

1 μl of the phage library at the appropriate dilution

Incubate the mixture at 37°C for 15 minutes to allow the phage to

11. Place the plates upside-down in a 37°C incubator. Plaques should be

HybriZAP®-2.1 Two-Hybrid Libraries 13